2024-03-28T15:10:07Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/85152019-08-30T11:32:38Zcom_10033_6832col_10033_6833
Panthel, Klaus
Jechlinger, Wolfgang
Matis, Alexander
Rohde, Manfred
Szostak, Michael
Lubitz, Werner
Haas, Rainer
2007-02-19T09:34:08Z
2003-01
2007-02-19T09:34:08Z
2003-01
Infection and Immunity 2003 71(1):109-116
0019-9567
1098-5522
12496155
10.1128/IAI.71.1.109-116.2003
http://hdl.handle.net/10033/8515
143412
en_US
American Society for Microbiology
Copyright © 2003, American Society for Microbiology
Generation of Helicobacter pylori Ghosts by PhiX Protein E-Mediated Inactivation and Their Evaluation as Vaccine Candidates
YES2018-06-13T04:21:10Zoai:repository.helmholtz-hzi.de:10033/86142019-08-30T11:32:39Zcom_10033_6832col_10033_6833
Rinas, U
Hellmuth, K
Kang, R
Seeger, A
Schlieker, H
2007-02-20T13:10:07Z
1995-12
2007-02-20T13:10:07Z
1995-12
Applied and Environmental Microbiology 1995 61(12):4147-4151
0099-2240
1098-5336
8534082
http://hdl.handle.net/10033/8614
167726
en_US
Entry of Escherichia coli into stationary phase is indicated by endogenous and exogenous accumulation of nucleobases.
YES2018-06-12T17:47:26Zoai:repository.helmholtz-hzi.de:10033/86282019-08-30T11:32:40Zcom_10033_6832col_10033_6833
Zogaj, Xhavit
Bokranz, Werner
Nimtz, Manfred
Römling, Ute
2007-02-20T13:26:19Z
2003-07
2007-02-20T13:26:19Z
2003-07
Infection and Immunity 2003 71(7):4151-4158
0019-9567
1098-5522
12819107
10.1128/IAI.71.7.4151-4158.2003
http://hdl.handle.net/10033/8628
162016
en_US
American Society for Microbiology
Copyright © 2003, American Society for Microbiology
Production of Cellulose and Curli Fimbriae by Members of the Family Enterobacteriaceae Isolated from the Human Gastrointestinal Tract
YES2018-06-13T05:29:34Zoai:repository.helmholtz-hzi.de:10033/85932019-08-30T11:37:44Zcom_10033_6832col_10033_6833
Kordel, M
Hofmann, B
Schomburg, D
Schmid, R D
2007-02-20T12:45:24Z
1991-08
2007-02-20T12:45:24Z
1991-08
Journal of Bacteriology 1991 173(15):4836-4841
0021-9193
1098-5530
1856176
http://hdl.handle.net/10033/8593
208163
Images
en_US
Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary X-ray diffraction data.
YES2018-06-13T00:40:28ZImagesoai:repository.helmholtz-hzi.de:10033/85992019-08-30T11:24:25Zcom_10033_6835com_10033_6832col_10033_6836
Li, Zu-Yi
Lang, Siegmund
Wagner, Fritz
Witte, Ludger
Wray, Victor
2007-02-20T12:51:47Z
1984-09
2007-02-20T12:51:47Z
1984-09
Applied and Environmental Microbiology 1984 48(3):610-617
0099-2240
1098-5336
http://hdl.handle.net/10033/8599
16346628
en_US
Copyright © 1984, American Society for Microbiology
Formation and Identification of Interfacial-Active Glycolipids from Resting Microbial Cells
YES2018-06-13T19:43:34Zoai:repository.helmholtz-hzi.de:10033/86732019-08-30T11:32:36Zcom_10033_6832col_10033_6833
Vallejo, Luis Felipe
Rinas, Ursula
2007-02-20T14:43:28Z
2004-09-02
2007-02-20T14:43:28Z
2004-09-02
Microbial Cell Factories 2004 3:11
1475-2859
15345063
10.1186/1475-2859-3-11
http://hdl.handle.net/10033/8673
517725
en_US
BioMed Central
http://www.microbialcellfactories.com/content/3/1/11
http://creativecommons.org/licenses/by/2.0
Copyright © 2004 Vallejo and Rinas; licensee BioMed Central Ltd.
Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins
YES2018-06-13T03:43:59Zoai:repository.helmholtz-hzi.de:10033/86902019-08-30T11:25:07Zcom_10033_6835com_10033_6832col_10033_6836
Strack, Dieter
Gross, Wiltrud
Wray, Victor
Grotjahn, Lutz
2007-02-21T08:28:25Z
1987-03
2007-02-21T08:28:25Z
1987-03
Plant Physiology 1987 83(3):475-478
0032-0889
1532-2548
http://hdl.handle.net/10033/8690
1056389
en_US
Enzymic Synthesis of Caffeoylglucaric Acid from Chlorogenic Acid and Glucaric Acid by a Protein Preparation from Tomato Cotyledons 1
YES2018-06-13T00:27:52Zoai:repository.helmholtz-hzi.de:10033/123442019-08-30T11:32:16Zcom_10033_6832col_10033_6833
JUDELE, Roxana
LASCHAT, SABINE
BARO, ANGELIKA
NIMTZ, MANFRED
2007-06-15T08:23:30Z
2007-06-15T08:23:30Z
2006-10-09
Tetrahedron 2006, 62:41
00404020
10.1016/j.tet.2006.07.087
http://hdl.handle.net/10033/12344
A series of unsubstituted and 1,4-disubstituted gallic catecholates 1, 6 and 7 as possible candidates for wedge-shaped mesogens were prepared starting from the respective benzene derivatives 2a–c and gallic esters 5a–h. The mesomorphic properties were investigated by DSC. However, only the 4,5-dinitro derivatives 1d,f–h with C8H17 and C10H21 to C12H25 alkyl side chains displayed mesophases, as evaluated by fluidity and optical anisotropy.
724574 bytes
application/pdf
YES
en_US
Elsevier Ltd
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6THR-4KPFKF1-9&_user=104184&_coverDate=10%2F09%2F2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000007378&_version=1&_urlVersion=0&_userid=104184&md5=e4d8aadacc0214f33c5e9e1e71ede965
Gallic Esters of 4,5-Dinitrocatechol as Potential Building Blocks for Thermotropic Liquid Crystals
Article2018-06-12T21:24:02ZA series of unsubstituted and 1,4-disubstituted gallic catecholates 1, 6 and 7 as possible candidates for wedge-shaped mesogens were prepared starting from the respective benzene derivatives 2a–c and gallic esters 5a–h. The mesomorphic properties were investigated by DSC. However, only the 4,5-dinitro derivatives 1d,f–h with C8H17 and C10H21 to C12H25 alkyl side chains displayed mesophases, as evaluated by fluidity and optical anisotropy.oai:repository.helmholtz-hzi.de:10033/123452019-08-30T11:32:13Zcom_10033_6832col_10033_6833
Heinz, Dirk W
Weiss, Manfred S
Wendt, K Ulrich
2007-06-15T11:09:24Z
2007-06-15T11:09:24Z
2006-01-01
Chembiochem 2006, 7(1):203-8
1439-4227
16317791
10.1002/cbic.200500459
http://hdl.handle.net/10033/12345
-1 bytes
application/pdf
YES
en
Wiley and Sons
Biomacromolecular interactions, assemblies and machines: a structural view.
Article2018-06-13T15:25:29Zoai:repository.helmholtz-hzi.de:10033/123562019-08-30T11:31:46Zcom_10033_6832col_10033_6833
Bösch, Andreas
Nimtz, Manfred
Mischnik, Petra
2007-06-19T12:34:55Z
2007-06-19T12:34:55Z
2007-06-19T12:34:55Z
Cellulose 2006, 13:4, 493-507
09690239,1572882X
10.1007/s10570-005-9029-9
http://hdl.handle.net/10033/12356
407488 bytes
application/pdf
YES
n/a
Springer Science+Business Media B.V.
Mechanistic studies on cationic ring-opening polymerisation of cyclodextrin derivatives using various Lewis acids
Article2018-06-12T23:41:19Zoai:repository.helmholtz-hzi.de:10033/124302019-08-30T11:30:58Zcom_10033_6832col_10033_6833
Hunke, Cornelia
Hirsch, Tatjana
Eichler, Jutta
2007-06-25T14:52:40Z
2007-06-25T14:52:40Z
2006-08-01
Chembiochem 2006, 7(8):1258-64
1439-4227
16810654
10.1002/cbic.200500465
http://hdl.handle.net/10033/12430
The Mena EVH1 domain, a protein-interaction module involved in actin-based cell motility, recognizes proline-rich ligand motifs, which are also present in the sequence of the surface protein ActA of Listeria monocytogenes. The interaction of ActA with host Mena EVH1 enables the bacterium to actively recruit host actin in order to spread into neighboring cells. Based on the crystal structure of Mena EVH1 in complex with a polyproline peptide ligand, we have generated a range of assembled peptides presenting the Mena EVH1 fragments that make up its discontinuous binding site for proline-rich ligands. Some of these peptides were found to inhibit the interaction of Mena EVH1 with the ligand pGolemi. One of them was further characterized at the level of individual amino acid residues; this yielded information on the contribution of individual positions of the peptides to the interaction with the ligand and identified sites for future structure optimization.
445080 bytes
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en
Structure-based synthetic mimicry of discontinuous protein binding sites: inhibitors of the interaction of Mena EVH1 domain with proline-rich ligands.
Article2018-06-12T17:43:14ZThe Mena EVH1 domain, a protein-interaction module involved in actin-based cell motility, recognizes proline-rich ligand motifs, which are also present in the sequence of the surface protein ActA of Listeria monocytogenes. The interaction of ActA with host Mena EVH1 enables the bacterium to actively recruit host actin in order to spread into neighboring cells. Based on the crystal structure of Mena EVH1 in complex with a polyproline peptide ligand, we have generated a range of assembled peptides presenting the Mena EVH1 fragments that make up its discontinuous binding site for proline-rich ligands. Some of these peptides were found to inhibit the interaction of Mena EVH1 with the ligand pGolemi. One of them was further characterized at the level of individual amino acid residues; this yielded information on the contribution of individual positions of the peptides to the interaction with the ligand and identified sites for future structure optimization.oai:repository.helmholtz-hzi.de:10033/124892019-08-30T11:25:43Zcom_10033_6832col_10033_6833
Wang, X
Rochon, M
Lamprokostopoulou, A
Lünsdorf, H
Nimtz, M
Römling, U
2007-07-02T08:59:50Z
2007-07-02T08:59:50Z
2006-10-01
Cell. Mol. Life Sci. 2006, 63(19-20):2352-63
1420-682X
16952050
10.1007/s00018-006-6222-4
http://hdl.handle.net/10033/12489
Commensal Escherichia coli form biofilms at body temperature by expressing the extracellular matrix components curli fimbriae and cellulose. The role of curli fimbriae and cellulose in the interaction of commensal E. coli with the intestinal epithelial cell line HT-29 was investigated. Expression of curli fimbriae by the typical commensal isolate E. coli TOB1 caused adherence and internalization of the bacteria and triggered IL-8 production in HT-29 cells. In particular, induction of IL-8 production was complex and involved curli-bound flagellin. While cellulose alone had no effect on the interaction of TOB1 with HT-29 cells, co-expression of cellulose with curli fimbriae decreased adherence to, internalization and IL-8 induction of HT-29 cells. Investigation of a panel of commensal isolates showed a partial correlation between expression of curli fimbriae and enhanced internalization and IL-8 production. In addition, a high immunostimulatory flagellin was identified. Thus, the consequences of expression of extracellular matrix components on commensal bacterial-host interactions are complex.
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en
Impact of biofilm matrix components on interaction of commensal Escherichia coli with the gastrointestinal cell line HT-29.
Article2018-06-13T00:28:59ZCommensal Escherichia coli form biofilms at body temperature by expressing the extracellular matrix components curli fimbriae and cellulose. The role of curli fimbriae and cellulose in the interaction of commensal E. coli with the intestinal epithelial cell line HT-29 was investigated. Expression of curli fimbriae by the typical commensal isolate E. coli TOB1 caused adherence and internalization of the bacteria and triggered IL-8 production in HT-29 cells. In particular, induction of IL-8 production was complex and involved curli-bound flagellin. While cellulose alone had no effect on the interaction of TOB1 with HT-29 cells, co-expression of cellulose with curli fimbriae decreased adherence to, internalization and IL-8 induction of HT-29 cells. Investigation of a panel of commensal isolates showed a partial correlation between expression of curli fimbriae and enhanced internalization and IL-8 production. In addition, a high immunostimulatory flagellin was identified. Thus, the consequences of expression of extracellular matrix components on commensal bacterial-host interactions are complex.oai:repository.helmholtz-hzi.de:10033/137212019-08-30T11:33:02Zcom_10033_6832col_10033_6833
LANGER, Olaf
Palme, Olov
Wray, Victor
Tokuda, Harukuni
Lang, Sigmund
2007-09-20T09:13:47Z
2007-09-20T09:13:47Z
2007-09-20T09:13:47Z
Process Biochemistry 2006, 41:10
00329592
10.1016/j.procbio.2006.07.036
http://hdl.handle.net/10033/13721
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n/a
Production and modification of bioactive surfactants
Article2018-06-13T20:02:46Zoai:repository.helmholtz-hzi.de:10033/146312019-08-30T11:34:48Zcom_10033_6832col_10033_6833
Seibel, Jürgen
Moraru, Roxana
Götze, Sven
Buchholz, Klaus
Na'amnieh, Shukrallah
Pawlowski, Alice
Hecht, Hans-Jürgen
2007-11-19T15:29:22Z
2007-11-19T15:29:22Z
2006-10-16
Carbohydr. Res. 2006, 341(14):2335-49
0008-6215
16870166
10.1016/j.carres.2006.07.001
http://hdl.handle.net/10033/14631
In the present study, we have coupled detailed acceptor and donor substrate studies of the fructosyltransferase (FTF, levansucrase) (EC 2.4.1.162) from Bacillus subtilis NCIMB 11871, with a structural model of the substrate enzyme complex in order to investigate in detail the roles of the active site amino acids in the catalytic action of the enzyme and the scope and limitation of substrates. Therefore we have isolated the ftf gene, expressed in Escherichia coli, yielding a levansucrase. Consequently, detailed acceptor property effects in the fructosylation by systematic variation of glycoside acceptors with respect to the positions (2, 3, 4 and 6) of the hydroxyl groups from equatorial to axial have been studied for preparative scale production of new oligosaccharides. Such investigations provided mechanistic insights of the FTF reaction. The configuration and the presence of the C-2 and C-3 hydroxyl groups of the glucopyranoside derivatives either as substrates or acceptors have been identified to be rate limiting for the trans-fructosylation process. The rates are rationalized on the basis of the coordination of d-glycopyranoside residues in (4)C(1) conformation with a network of amino acids by Arg360, Tyr411, Glu342, Trp85, Asp247 and Arg246 stabilization of both acceptors and substrates. In addition we also describe the first FTF reaction, which catalyzes the beta-(1-->2)-fructosyl transfer to 2-OH of L-sugars (L-glucose, L-rhamnose, L-galactose, L-fucose, L-xylose) presumably in a (1)C(4) conformation. In those conformations, the L-glycopyranosides are stabilized by the same hydrogen network. Structures of the acceptor products were determined by NMR and mass spectrometry analysis.
1035891 bytes
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en
Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase).
Article2018-06-13T01:05:48ZIn the present study, we have coupled detailed acceptor and donor substrate studies of the fructosyltransferase (FTF, levansucrase) (EC 2.4.1.162) from Bacillus subtilis NCIMB 11871, with a structural model of the substrate enzyme complex in order to investigate in detail the roles of the active site amino acids in the catalytic action of the enzyme and the scope and limitation of substrates. Therefore we have isolated the ftf gene, expressed in Escherichia coli, yielding a levansucrase. Consequently, detailed acceptor property effects in the fructosylation by systematic variation of glycoside acceptors with respect to the positions (2, 3, 4 and 6) of the hydroxyl groups from equatorial to axial have been studied for preparative scale production of new oligosaccharides. Such investigations provided mechanistic insights of the FTF reaction. The configuration and the presence of the C-2 and C-3 hydroxyl groups of the glucopyranoside derivatives either as substrates or acceptors have been identified to be rate limiting for the trans-fructosylation process. The rates are rationalized on the basis of the coordination of d-glycopyranoside residues in (4)C(1) conformation with a network of amino acids by Arg360, Tyr411, Glu342, Trp85, Asp247 and Arg246 stabilization of both acceptors and substrates. In addition we also describe the first FTF reaction, which catalyzes the beta-(1-->2)-fructosyl transfer to 2-OH of L-sugars (L-glucose, L-rhamnose, L-galactose, L-fucose, L-xylose) presumably in a (1)C(4) conformation. In those conformations, the L-glycopyranosides are stabilized by the same hydrogen network. Structures of the acceptor products were determined by NMR and mass spectrometry analysis.oai:repository.helmholtz-hzi.de:10033/147332019-08-30T11:34:48Zcom_10033_6832col_10033_6833
Schulze, Jörg O
Schubert, Wolf-Dieter
Moser, Jürgen
Jahn, Dieter
Heinz, Dirk W
2007-11-26T12:54:19Z
2007-11-26T12:54:19Z
2006-05-19
J. Mol. Biol. 2006, 358(5):1212-20
0022-2836
16564539
10.1016/j.jmb.2006.02.064
http://hdl.handle.net/10033/14733
Glutamate-1-semialdehyde 2,1-aminomutase (GSAM) is the second enzyme in the C(5) pathway of tetrapyrrole biosynthesis found in most bacteria, in archaea and in plants. It catalyzes the transamination of glutamate-1-semialdehyde to 5-aminolevulinic acid (ALA) in a pyridoxal 5'-phosphate (PLP)-dependent manner. We present the crystal structure of GSAM from the thermophilic cyanobacterium Thermosynechococcus elongatus (GSAM(Tel)) in its PLP-bound form at 2.85A resolution. GSAM(Tel) is a symmetric homodimer, whereas GSAM from Synechococcus (GSAM(Syn)) has been described as asymmetric. The symmetry of GSAM(Tel) thus challenges the previously proposed negative cooperativity between monomers of this enzyme. Furthermore, GSAM(Tel) reveals an extensive flexible region at the interface of the proposed complex of GSAM with glutamyl-tRNA reductase (GluTR), the preceding enzyme in tetrapyrrole biosynthesis. Compared to GSAM(Syn), the monomers of GSAM(Tel) are rotated away from each other along the dimerization interface by 10 degrees . The associated flexibility of GSAM may be essential for complex formation with GluTR to occur. Unexpectedly, we find that GSAM is structurally related to 5-aminolevulinate synthase (ALAS), the ALA-producing enzyme in the Shemin pathway of alpha-proteobacteria and non-plant eukaryotes. This structural relationship applies also to the corresponding subfamilies of PLP-dependent enzymes. We thus propose that the CoA-subfamily (including ALAS) and the aminotransferase subfamily II (including GSAM) are evolutionarily closely related and that ALAS may thus have evolved from GSAM.
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en
Evolutionary relationship between initial enzymes of tetrapyrrole biosynthesis.
Article2018-06-13T09:18:33ZGlutamate-1-semialdehyde 2,1-aminomutase (GSAM) is the second enzyme in the C(5) pathway of tetrapyrrole biosynthesis found in most bacteria, in archaea and in plants. It catalyzes the transamination of glutamate-1-semialdehyde to 5-aminolevulinic acid (ALA) in a pyridoxal 5'-phosphate (PLP)-dependent manner. We present the crystal structure of GSAM from the thermophilic cyanobacterium Thermosynechococcus elongatus (GSAM(Tel)) in its PLP-bound form at 2.85A resolution. GSAM(Tel) is a symmetric homodimer, whereas GSAM from Synechococcus (GSAM(Syn)) has been described as asymmetric. The symmetry of GSAM(Tel) thus challenges the previously proposed negative cooperativity between monomers of this enzyme. Furthermore, GSAM(Tel) reveals an extensive flexible region at the interface of the proposed complex of GSAM with glutamyl-tRNA reductase (GluTR), the preceding enzyme in tetrapyrrole biosynthesis. Compared to GSAM(Syn), the monomers of GSAM(Tel) are rotated away from each other along the dimerization interface by 10 degrees . The associated flexibility of GSAM may be essential for complex formation with GluTR to occur. Unexpectedly, we find that GSAM is structurally related to 5-aminolevulinate synthase (ALAS), the ALA-producing enzyme in the Shemin pathway of alpha-proteobacteria and non-plant eukaryotes. This structural relationship applies also to the corresponding subfamilies of PLP-dependent enzymes. We thus propose that the CoA-subfamily (including ALAS) and the aminotransferase subfamily II (including GSAM) are evolutionarily closely related and that ALAS may thus have evolved from GSAM.oai:repository.helmholtz-hzi.de:10033/152302019-08-30T11:34:48Zcom_10033_6832col_10033_6833
Schulze, Jörg O
Masoumi, Ava
Nickel, Daniel
Jahn, Martina
Jahn, Dieter
Schubert, Wolf-Dieter
Heinz, Dirk W
Division of Structural Biology, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
2007-12-14T15:02:09Z
2007-12-14T15:02:09Z
2006-09-01
Crystal structure of a non-discriminating glutamyl-tRNA synthetase. 2006, 361 (5):888-97 J. Mol. Biol.
0022-2836
16876193
10.1016/j.jmb.2006.06.054
http://hdl.handle.net/10033/15230
Journal of molecular biology
Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.
en
Amino Acid Sequence
Anticodon
Binding Sites
Catalysis
Crystallography, X-Ray
Cyanobacteria
Glutamate-tRNA Ligase
Glutamic Acid
Models, Molecular
Molecular Sequence Data
Protein Binding
Protein Structure, Tertiary
Crystal structure of a non-discriminating glutamyl-tRNA synthetase.
Article2018-06-12T23:29:03ZError-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.oai:repository.helmholtz-hzi.de:10033/157182019-08-30T11:35:39Zcom_10033_6832col_10033_6833
Galeyeva, Yana
Helbig, Sarah
Morr, Michael
Sasse, Florenz
Nimtz, Manfred
Laschat, Sabine
Baro, Angelika
Institut für Organische Chemie, Universität Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart.
2008-01-04T14:51:08Z
2008-01-04T14:51:08Z
2006-08
Total synthesis and biological evaluation of (-)-pectinatone employing a methyl-branched wax ester as key building block. 2006, 3 (8):935-41 Chem. Biodivers.
1612-1880
17193325
10.1002/cbdv.200690096
http://hdl.handle.net/10033/15718
Chemistry & biodiversity
Unnatural (-)-pectinatone ((-)-3) was prepared in five steps starting from the highly methyl-branched wax ester 4, employing bromination of the ester enolate and subsequent base-induced elimination to the enoate 6 as the key step. Both (-)-3 and the amides 8b and 8c, which were isolated as by-products in the reaction sequence, displayed antimicrobial activity and cytotoxicity.
en
Animals
Anti-Bacterial Agents
Biological Products
Cell Line
Escherichia coli
Esters
Mice
Micrococcus luteus
Molecular Structure
Pyrones
Staphylococcus aureus
Waxes
Total synthesis and biological evaluation of (-)-pectinatone employing a methyl-branched wax ester as key building block.
Article2018-06-12T23:50:15ZUnnatural (-)-pectinatone ((-)-3) was prepared in five steps starting from the highly methyl-branched wax ester 4, employing bromination of the ester enolate and subsequent base-induced elimination to the enoate 6 as the key step. Both (-)-3 and the amides 8b and 8c, which were isolated as by-products in the reaction sequence, displayed antimicrobial activity and cytotoxicity.oai:repository.helmholtz-hzi.de:10033/157372019-08-30T11:33:03Zcom_10033_6832col_10033_6833
el-Enshasy, Hesham
Kleine, Joachim
Rinas, Ursula
Helmholtz zENTRUM FÜR iNFEKTIONSFORSCHUNG
2008-01-07T11:44:27Z
2008-01-07T11:44:27Z
2006-10
00329592
10.1016/j.procbio.2006.05.024
http://hdl.handle.net/10033/15737
Process Biochemistry8(2006); 41; pp.2103-2112
Agitation effects on morphology and protein productive fractions of filamentous and pelleted growth forms of recombinant Aspergillus niger
Article2018-06-12T22:37:24Zoai:repository.helmholtz-hzi.de:10033/158362019-08-30T11:25:11Zcom_10033_6832col_10033_6833
Gueorguieva, Ludmila
Vallejo, Luis Felipe
Rinas, Ursula
Seidel-Morgenstern, Andreas
Otto-von-Guericke-Universität Magdeburg, Institut für Verfahrenstechnik, PO Box 4120, D-39106 Magdeburg, Germany.
2008-01-08T15:15:34Z
2008-01-08T15:15:34Z
2006-12-01
Discontinuous and continuous separation of the monomeric and dimeric forms of human bone morphogenetic protein-2 from renaturation batches. 2006, 1135 (2):142-50notJ Chromatogr A
0021-9673
17064713
10.1016/j.chroma.2006.08.061
http://hdl.handle.net/10033/15836
Journal of chromatography. A
Bone morphogenetic protein-2 (BMP-2) is one of the most interesting of the approximately 14 BMPs which belong to the transforming-growth-factor-beta (TGF-beta) superfamily. BMP-2 induces bone formation and thus plays an important role as a pharmaceutical protein. Recently, rhBMP-2 has been produced in form of inactive inclusion bodies in Escherichia coli. After solubilization and renaturation the biologically active dimeric form of rhBMP-2 can be generated. However, inactive monomers of BMP-2 are also formed during the renaturation process which must be separated from the active dimeric BMP-2. The purpose of this paper is to present: (a) results of an experimental study of a chromatographic separation of the monomeric and dimeric forms; and (b) a concept for a continuous counter-current simulated moving bed (SMB) process. The capacity of heparin as stationary phase was estimated for different salt concentrations in the mobile phase. A simulation study of a three-zone SMB process was performed applying a two step salt gradient. The results reveal the potential of the process for the purification of the dimeric BMP-2.
en
Bone Morphogenetic Proteins
Chromatography, Liquid
Dimerization
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Heparin
Protein Renaturation
Recombinant Proteins
Spectrophotometry, Ultraviolet
Transforming Growth Factor beta
Discontinuous and continuous separation of the monomeric and dimeric forms of human bone morphogenetic protein-2 from renaturation batches.
Article2018-06-12T16:47:54ZBone morphogenetic protein-2 (BMP-2) is one of the most interesting of the approximately 14 BMPs which belong to the transforming-growth-factor-beta (TGF-beta) superfamily. BMP-2 induces bone formation and thus plays an important role as a pharmaceutical protein. Recently, rhBMP-2 has been produced in form of inactive inclusion bodies in Escherichia coli. After solubilization and renaturation the biologically active dimeric form of rhBMP-2 can be generated. However, inactive monomers of BMP-2 are also formed during the renaturation process which must be separated from the active dimeric BMP-2. The purpose of this paper is to present: (a) results of an experimental study of a chromatographic separation of the monomeric and dimeric forms; and (b) a concept for a continuous counter-current simulated moving bed (SMB) process. The capacity of heparin as stationary phase was estimated for different salt concentrations in the mobile phase. A simulation study of a three-zone SMB process was performed applying a two step salt gradient. The results reveal the potential of the process for the purification of the dimeric BMP-2.oai:repository.helmholtz-hzi.de:10033/173322019-08-30T11:33:01Zcom_10033_6832col_10033_6833
Jerz, Gerold
Arrey, Tabiwang N.
Wray, Victor
Du, Qizhen
Winterhalter, Peter
Helmholtz Zentrum für Infektionsforschung GmbH
2008-02-01T08:32:13Z
2008-02-01T08:32:13Z
2007
14668564
10.1016/j.ifset.2007.03.024
http://hdl.handle.net/10033/17332
Innovative Food Science & Emerging Technologies
Elsevier Science
Structural characterization of 13(2)-hydroxy-(13(2)-S)-phaeophytin-a from leaves and stems of Amaranthus tricolor isolated by high-speed countercurrent chromatography
Article2018-06-12T21:38:16Zoai:repository.helmholtz-hzi.de:10033/185922019-08-30T11:32:37Zcom_10033_6835com_10033_6832col_10033_6836
Gutzeit, D.
Wray, Victor
Winterhalter, P.
Jerz, Gerold
Institute of Food Chemistry, Technical University of Braunschweig
2008-02-18T09:58:22Z
2008-02-18T09:58:22Z
2007
(2007) Chromatographia 65 (1-2); pp. 1-7
00095893
16121112
10.1365/s10337-006-0105-6
http://hdl.handle.net/10033/18592
Chromatographia 65 (1-2); pp. 1-7
null
Springer Science Business Media
Preparative Isolation and Purification of Flavonoids and Protocatechuic Acid from Sea Buckthorn Juice Concentrate (Hippophae rhamnoides) by High-Speed Counter-Current Chromatography
Article2018-06-13T02:41:14Zoai:repository.helmholtz-hzi.de:10033/188252019-08-30T11:34:48Zcom_10033_6832col_10033_6833
Zieseniss, Anke
Schroeder, Ulrich
Buchmeier, Sabine
Schoenenberger, Cora-Ann
van den Heuvel, Joop
Jockusch, Brigitte M
Illenberger, Susanne
Cell Biology, Zoological Institute, Technical University of Braunschweig, Biocentre, Spielmannstrasse 7, 38092 Braunschweig, Germany.
2008-02-21T13:41:29Z
2008-02-21T13:41:29Z
2007-03
Raver1 is an integral component of muscle contractile elements. 2007, 327 (3):583-94 Cell Tissue Res.
0302-766X
17096167
10.1007/s00441-006-0322-1
http://hdl.handle.net/10033/18825
Cell and tissue research
Raver1, a ubiquitously expressed protein, was originally identified as a ligand for metavinculin, the muscle-specific isoform of the microfilament-associated protein vinculin. The protein resides primarily in the nucleus, where it colocalises and may interact with polypyrimidine-tract-binding protein, which is involved in alternative splicing processes. During skeletal muscle differentiation, raver1 translocates to the cytoplasm and eventually targets the Z-line of sarcomeres. Here, it colocalises with metavinculin, vinculin and alpha-actinin, all of which have biochemically been identified as raver1 ligands. To obtain more information about the potential role of raver1 in muscle structure and function, we have investigated its distribution and fine localisation in mouse striated and smooth muscle, by using three monoclonal antibodies that recognise epitopes in different regions of the raver1 protein. Our immunofluorescence and immunoelectron-microscopic results indicate that the cytoplasmic accumulation of raver1 is not confined to skeletal muscle but also occurs in heart and smooth muscle. Unlike vinculin and metavinculin, cytoplasmic raver1 is not restricted to costameres but additionally represents an integral part of the sarcomere. In isolated myofibrils and in ultrathin sections of skeletal muscle, raver1 has been found concentrated at the I-Z-I band. A minor fraction of raver1 is present in the nuclei of all three types of muscle. These data indicate that, during muscle differentiation, raver1 might link gene expression with structural functions of the contractile machinery of muscle.
en
Actins
Animals
Carrier Proteins
Electrophoresis, Polyacrylamide Gel
Mice
Microscopy, Immunoelectron
Muscle Contraction
Muscle, Skeletal
Muscle, Smooth
Nuclear Proteins
Sarcomeres
Vinculin
Raver1 is an integral component of muscle contractile elements.
Article2018-06-12T23:19:26ZRaver1, a ubiquitously expressed protein, was originally identified as a ligand for metavinculin, the muscle-specific isoform of the microfilament-associated protein vinculin. The protein resides primarily in the nucleus, where it colocalises and may interact with polypyrimidine-tract-binding protein, which is involved in alternative splicing processes. During skeletal muscle differentiation, raver1 translocates to the cytoplasm and eventually targets the Z-line of sarcomeres. Here, it colocalises with metavinculin, vinculin and alpha-actinin, all of which have biochemically been identified as raver1 ligands. To obtain more information about the potential role of raver1 in muscle structure and function, we have investigated its distribution and fine localisation in mouse striated and smooth muscle, by using three monoclonal antibodies that recognise epitopes in different regions of the raver1 protein. Our immunofluorescence and immunoelectron-microscopic results indicate that the cytoplasmic accumulation of raver1 is not confined to skeletal muscle but also occurs in heart and smooth muscle. Unlike vinculin and metavinculin, cytoplasmic raver1 is not restricted to costameres but additionally represents an integral part of the sarcomere. In isolated myofibrils and in ultrathin sections of skeletal muscle, raver1 has been found concentrated at the I-Z-I band. A minor fraction of raver1 is present in the nuclei of all three types of muscle. These data indicate that, during muscle differentiation, raver1 might link gene expression with structural functions of the contractile machinery of muscle.oai:repository.helmholtz-hzi.de:10033/196732019-08-30T11:30:32Zcom_10033_6832col_10033_6833
Ganzlin, Markus
Marose, Stefan
Lu, Xin
Hitzmann, Bernd
Scheper, Thomas
Rinas, Ursula
Helmholtz Centre for Infection Research (former German Research Centre for Biotechnology - GBF), Inhoffenstr. 7, 38124 Braunschweig, Germany.
2008-03-04T14:26:26Z
2008-03-04T14:26:26Z
2007-12-01
In situ multi-wavelength fluorescence spectroscopy as effective tool to simultaneously monitor spore germination, metabolic activity and quantitative protein production in recombinant Aspergillus niger fed-batch cultures. 2007, 132 (4):461-8 J. Biotechnol.
0168-1656
17905460
10.1016/j.jbiotec.2007.08.032
http://hdl.handle.net/10033/19673
Journal of biotechnology
The production of a mutant green fluorescent protein (S65TGFP), controlled by the maltose inducible glucoamylase promoter, was followed in situ in fed-batch cultures of recombinant Aspergillus niger using multi-wavelength fluorescence spectroscopy. Disturbance of quantitative product analysis by interfering fluorescence signals was resolved by using a set of defined combinations of excitation and emission wavelengths (lambda(ex)/lambda(em)). This technique resulted in excellent linearity between on-line signal and off-line determined S65TGFP concentrations. Spore germination was detectable in situ by monitoring the back scattered light intensity. Moreover, flavin-like fluorophores were identified as the dominating fungal host fluorophores. The time-dependent intensity of this fluorophore, potentially fungal flavin-containing oxidoreductase(s), did not correlate with the biomass concentration but correlated well with the fungal metabolic activity (e.g. respiratory activity). Other fluorophores commonly found in microbial cultures such NADH, pyridoxine and the aromatic amino acids, tryptophan, phenylalanine and tyrosine did not contribute significantly to the culture fluorescence of A. niger. Thus, multi-wavelength fluorescence spectroscopy has proven to be an effective tool for simultaneous on-line monitoring of the most relevant process variables in fungal cultures, e.g. spore germination, metabolic activity, and quantitative product formation.
en
Aspergillus niger
Bioreactors
Germination
Green Fluorescent Proteins
Industrial Microbiology
Spectrometry, Fluorescence
Spores, Fungal
In situ multi-wavelength fluorescence spectroscopy as effective tool to simultaneously monitor spore germination, metabolic activity and quantitative protein production in recombinant Aspergillus niger fed-batch cultures.
Article2018-06-13T15:29:51ZThe production of a mutant green fluorescent protein (S65TGFP), controlled by the maltose inducible glucoamylase promoter, was followed in situ in fed-batch cultures of recombinant Aspergillus niger using multi-wavelength fluorescence spectroscopy. Disturbance of quantitative product analysis by interfering fluorescence signals was resolved by using a set of defined combinations of excitation and emission wavelengths (lambda(ex)/lambda(em)). This technique resulted in excellent linearity between on-line signal and off-line determined S65TGFP concentrations. Spore germination was detectable in situ by monitoring the back scattered light intensity. Moreover, flavin-like fluorophores were identified as the dominating fungal host fluorophores. The time-dependent intensity of this fluorophore, potentially fungal flavin-containing oxidoreductase(s), did not correlate with the biomass concentration but correlated well with the fungal metabolic activity (e.g. respiratory activity). Other fluorophores commonly found in microbial cultures such NADH, pyridoxine and the aromatic amino acids, tryptophan, phenylalanine and tyrosine did not contribute significantly to the culture fluorescence of A. niger. Thus, multi-wavelength fluorescence spectroscopy has proven to be an effective tool for simultaneous on-line monitoring of the most relevant process variables in fungal cultures, e.g. spore germination, metabolic activity, and quantitative product formation.oai:repository.helmholtz-hzi.de:10033/196742019-08-30T11:37:44Zcom_10033_6832col_10033_6833
Rinas, Ursula
Hoffmann, Frank
Betiku, Eriola
Estapé, David
Marten, Sabine
Biochemical Engineering Division, GBF German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. URI@gbf.de
2008-03-04T14:38:17Z
2008-03-04T14:38:17Z
2007-01-01
Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli. 2007, 127 (2):244-57 J. Biotechnol.
0168-1656
16945443
10.1016/j.jbiotec.2006.07.004
http://hdl.handle.net/10033/19674
Journal of biotechnology
During production in recombinant Escherichia coli, the human basic fibroblast growth factor (hFGF-2) partly aggregates into stable cytoplasmic inclusion bodies. These inclusion bodies additionally contain significant amounts of the heat-shock chaperone DnaK, and putative DnaK substrates such as the elongation factor Tu (ET-Tu) and the metabolic enzymes dihydrolipoamide dehydrogenase (LpdA), tryptophanase (TnaA), and d-tagatose-1,6-bisphosphate aldolase (GatY). Guanidinium hydrochloride induced disaggregation studies carried out in vitro on artificial aggregates generated through thermal aggregation of purified hFGF-2 revealed identical disaggregation profiles as hFGF-2 inclusion bodies indicating that the heterogenic composition of inclusion bodies did not influence the strength of interactions of hFGF-2 in aggregates formed in vivo as inclusion bodies compared to those generated in vitro from native and pure hFGF-2 through thermal aggregation. Compared to unfolding of native hFGF-2, higher concentrations of denaturant were required to dissolve hFGF-2 aggregates showing that more energy is required for disruption of interactions in both types of protein aggregates compared to the unfolding of the native protein. In vivo dissolution of hFGF-2 inclusion bodies was studied through coexpression of chaperones of the DnaK and GroEL family and ClpB and combinations thereof. None of the chaperone combinations was able to completely prevent the initial formation of inclusion bodies, but upon prolonged incubation mediated disaggregation of otherwise stable inclusion bodies. The GroEL system was particularly efficient in inclusion body dissolution but did not lead to a corresponding increase in soluble hFGF-2 rather was promoting the proteolysis of the recombinant growth factor. Coproduction of the disaggregating DnaK system and ClpB in conjunction with small amounts of the chaperonins GroELS was most efficient in disaggregation with concomitant formation of soluble hFGF-2. Thus, fine-balanced coproduction of chaperone combinations can play an important role in the production of soluble recombinant proteins with a high aggregation propensity not through prevention of aggregation but predominantly through their disaggregating properties.
en
Escherichia coli
Escherichia coli Proteins
Fibroblast Growth Factor 2
GroEL Protein
GroES Protein
HSP40 Heat-Shock Proteins
HSP70 Heat-Shock Proteins
Humans
Inclusion Bodies
Molecular Chaperones
Mutation
Protein Denaturation
Protein Folding
Protein Structure, Quaternary
Proteome
Recombinant Proteins
Solubility
Thermodynamics
Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli.
Article2018-06-13T03:43:30ZDuring production in recombinant Escherichia coli, the human basic fibroblast growth factor (hFGF-2) partly aggregates into stable cytoplasmic inclusion bodies. These inclusion bodies additionally contain significant amounts of the heat-shock chaperone DnaK, and putative DnaK substrates such as the elongation factor Tu (ET-Tu) and the metabolic enzymes dihydrolipoamide dehydrogenase (LpdA), tryptophanase (TnaA), and d-tagatose-1,6-bisphosphate aldolase (GatY). Guanidinium hydrochloride induced disaggregation studies carried out in vitro on artificial aggregates generated through thermal aggregation of purified hFGF-2 revealed identical disaggregation profiles as hFGF-2 inclusion bodies indicating that the heterogenic composition of inclusion bodies did not influence the strength of interactions of hFGF-2 in aggregates formed in vivo as inclusion bodies compared to those generated in vitro from native and pure hFGF-2 through thermal aggregation. Compared to unfolding of native hFGF-2, higher concentrations of denaturant were required to dissolve hFGF-2 aggregates showing that more energy is required for disruption of interactions in both types of protein aggregates compared to the unfolding of the native protein. In vivo dissolution of hFGF-2 inclusion bodies was studied through coexpression of chaperones of the DnaK and GroEL family and ClpB and combinations thereof. None of the chaperone combinations was able to completely prevent the initial formation of inclusion bodies, but upon prolonged incubation mediated disaggregation of otherwise stable inclusion bodies. The GroEL system was particularly efficient in inclusion body dissolution but did not lead to a corresponding increase in soluble hFGF-2 rather was promoting the proteolysis of the recombinant growth factor. Coproduction of the disaggregating DnaK system and ClpB in conjunction with small amounts of the chaperonins GroELS was most efficient in disaggregation with concomitant formation of soluble hFGF-2. Thus, fine-balanced coproduction of chaperone combinations can play an important role in the production of soluble recombinant proteins with a high aggregation propensity not through prevention of aggregation but predominantly through their disaggregating properties.oai:repository.helmholtz-hzi.de:10033/197582019-08-30T11:37:23Zcom_10033_6832col_10033_6833
Hagelueken, Gregor
Wiehlmann, Lutz
Adams, Thorsten M
Kolmar, Harald
Heinz, Dirk W
Tümmler, Burkhard
Schubert, Wolf-Dieter
Molecular Host-Pathogen Interactions, Division of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.
2008-03-05T10:23:42Z
2008-03-05T10:23:42Z
2007-07-24
Crystal structure of the electron transfer complex rubredoxin rubredoxin reductase of Pseudomonas aeruginosa. 2007, 104 (30):12276-81 Proc. Natl. Acad. Sci. U.S.A.
0027-8424
17636129
10.1073/pnas.0702919104
http://hdl.handle.net/10033/19758
Proceedings of the National Academy of Sciences of the United States of America
Crude oil spills represent a major ecological threat because of the chemical inertness of the constituent n-alkanes. The Gram-negative bacterium Pseudomonas aeruginosa is one of the few bacterial species able to metabolize such compounds. Three chromosomal genes, rubB, rubA1, and rubA2 coding for an NAD(P)H:rubredoxin reductase (RdxR) and two rubredoxins (Rdxs) are indispensable for this ability. They constitute an electron transport (ET) pathway that shuttles reducing equivalents from carbon metabolism to the membrane-bound alkane hydroxylases AlkB1 and AlkB2. The RdxR-Rdx system also is crucial as part of the oxidative stress response in archaea or anaerobic bacteria. The redox couple has been analyzed in detail as a model system for ET processes. We have solved the structure of RdxR of P. aeruginosa both alone and in complex with Rdx, without the need for cross-linking, and both structures were refined at 2.40- and 2.45-A resolution, respectively. RdxR consists of two cofactor-binding domains and a C-terminal domain essential for the specific recognition of Rdx. Only a small number of direct interactions govern mutual recognition of RdxR and Rdx, corroborating the transient nature of the complex. The shortest distance between the redox centers is observed to be 6.2 A.
en
Alkanes
Binding Sites
Crystallography, X-Ray
Dimerization
Electrons
Electrostatics
Flavin-Adenine Dinucleotide
Models, Molecular
NADH, NADPH Oxidoreductases
NADP
Nickel
Oxidation-Reduction
Phylogeny
Protein Binding
Protein Structure, Quaternary
Protein Structure, Tertiary
Pseudomonas aeruginosa
Rubredoxins
Crystal structure of the electron transfer complex rubredoxin rubredoxin reductase of Pseudomonas aeruginosa.
Article2018-06-13T07:24:00ZCrude oil spills represent a major ecological threat because of the chemical inertness of the constituent n-alkanes. The Gram-negative bacterium Pseudomonas aeruginosa is one of the few bacterial species able to metabolize such compounds. Three chromosomal genes, rubB, rubA1, and rubA2 coding for an NAD(P)H:rubredoxin reductase (RdxR) and two rubredoxins (Rdxs) are indispensable for this ability. They constitute an electron transport (ET) pathway that shuttles reducing equivalents from carbon metabolism to the membrane-bound alkane hydroxylases AlkB1 and AlkB2. The RdxR-Rdx system also is crucial as part of the oxidative stress response in archaea or anaerobic bacteria. The redox couple has been analyzed in detail as a model system for ET processes. We have solved the structure of RdxR of P. aeruginosa both alone and in complex with Rdx, without the need for cross-linking, and both structures were refined at 2.40- and 2.45-A resolution, respectively. RdxR consists of two cofactor-binding domains and a C-terminal domain essential for the specific recognition of Rdx. Only a small number of direct interactions govern mutual recognition of RdxR and Rdx, corroborating the transient nature of the complex. The shortest distance between the redox centers is observed to be 6.2 A.oai:repository.helmholtz-hzi.de:10033/197742019-08-30T11:37:23Zcom_10033_6832col_10033_6833
Wollert, Thomas
Heinz, Dirk W
Schubert, Wolf-Dieter
Molecular Host-Pathogen Interactions, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.
2008-03-05T10:30:35Z
2008-03-05T10:30:35Z
2007-08-28
Thermodynamically reengineering the listerial invasion complex InlA/E-cadherin. 2007, 104 (35):13960-5 Proc. Natl. Acad. Sci. U.S.A.
0027-8424
17715295
10.1073/pnas.0702199104
http://hdl.handle.net/10033/19774
Proceedings of the National Academy of Sciences of the United States of America
Biological processes essentially all depend on the specific recognition between macromolecules and their interaction partners. Although many such interactions have been characterized both structurally and biophysically, the thermodynamic effects of small atomic changes remain poorly understood. Based on the crystal structure of the bacterial invasion protein internalin (InlA) of Listeria monocytogenes in complex with its human receptor E-cadherin (hEC1), we analyzed the interface to identify single amino acid substitutions in InlA that would potentially improve the overall quality of interaction and hence increase the weak binding affinity of the complex. Dissociation constants of InlA-variant/hEC1 complexes, as well as enthalpy and entropy of binding, were quantified by isothermal titration calorimetry. All single substitutions indeed significantly increase binding affinity. Structural changes were verified crystallographically at < or =2.0-A resolution, allowing thermodynamic characteristics of single substitutions to be rationalized structurally and providing unique insights into atomic contributions to binding enthalpy and entropy. Structural and thermodynamic data of all combinations of individual substitutions result in a thermodynamic network, allowing the source of cooperativity between distant recognition sites to be identified. One such pair of single substitutions improves affinity 5,000-fold. We thus demonstrate that rational reengineering of protein complexes is possible by making use of physically distant hot spots of recognition.
en
Amino Acid Substitution
Asparagine
Bacterial Proteins
Binding Sites
Cadherins
Calorimetry
Genetic Engineering
Glycine
Humans
Kinetics
Listeria monocytogenes
Models, Molecular
Protein Binding
Protein Conformation
Recombinant Proteins
Serine
Thermodynamics
Tyrosine
Variation (Genetics)
Thermodynamically reengineering the listerial invasion complex InlA/E-cadherin.
Article2018-06-13T03:54:24ZBiological processes essentially all depend on the specific recognition between macromolecules and their interaction partners. Although many such interactions have been characterized both structurally and biophysically, the thermodynamic effects of small atomic changes remain poorly understood. Based on the crystal structure of the bacterial invasion protein internalin (InlA) of Listeria monocytogenes in complex with its human receptor E-cadherin (hEC1), we analyzed the interface to identify single amino acid substitutions in InlA that would potentially improve the overall quality of interaction and hence increase the weak binding affinity of the complex. Dissociation constants of InlA-variant/hEC1 complexes, as well as enthalpy and entropy of binding, were quantified by isothermal titration calorimetry. All single substitutions indeed significantly increase binding affinity. Structural changes were verified crystallographically at < or =2.0-A resolution, allowing thermodynamic characteristics of single substitutions to be rationalized structurally and providing unique insights into atomic contributions to binding enthalpy and entropy. Structural and thermodynamic data of all combinations of individual substitutions result in a thermodynamic network, allowing the source of cooperativity between distant recognition sites to be identified. One such pair of single substitutions improves affinity 5,000-fold. We thus demonstrate that rational reengineering of protein complexes is possible by making use of physically distant hot spots of recognition.oai:repository.helmholtz-hzi.de:10033/229332019-08-30T11:37:23Zcom_10033_6835com_10033_6832col_10033_6836
Scheibner, M
Hülsdau, B
Zelena, K
Nimtz, M
de Boer, L
Berger, RG
Zorn, H
Zentrum Angewandte Chemie, Institut für Lebensmittelchemie der Universität Hannover, Wunstorfer Straße 14, 30453, Hannover, Germany.
2008-04-10T14:42:13Z
2008-04-10T14:42:13Z
2008-01
Novel peroxidases of Marasmius scorodonius degrade beta-carotene. 2008, 77 (6):1241-1250 Appl. Microbiol. Biotechnol.
0175-7598
18038130
10.1007/s00253-007-1261-9
http://hdl.handle.net/10033/22933
Applied microbiology and biotechnology
Two extracellular enzymes (MsP1 and MsP2) capable of efficient beta-carotene degradation were purified from culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). Under native conditions, the enzymes exhibited molecular masses of ~150 and ~120 kDa, respectively. SDS-PAGE and mass spectrometric data suggested a composition of two identical subunits for both enzymes. Biochemical characterisation of the purified proteins showed isoelectric points of 3.7 and 3.5, and the presence of heme groups in the active enzymes. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. cDNAs of 1,604 to 1,923 bp, containing open reading frames (ORF) of 508 to 513 amino acids, respectively, were cloned from a cDNA library of M. scorodonius. These data suggest glycosylation degrees of ~23% for MsP1 and 8% for MsP2. Databank homology searches revealed sequence homologies of MsP1 and MsP2 to unusual peroxidases of the fungi Thanatephorus cucumeris (DyP) and Termitomyces albuminosus (TAP).
ENG
Novel peroxidases of Marasmius scorodonius degrade beta-carotene.
Article2018-06-13T19:31:11ZTwo extracellular enzymes (MsP1 and MsP2) capable of efficient beta-carotene degradation were purified from culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). Under native conditions, the enzymes exhibited molecular masses of ~150 and ~120 kDa, respectively. SDS-PAGE and mass spectrometric data suggested a composition of two identical subunits for both enzymes. Biochemical characterisation of the purified proteins showed isoelectric points of 3.7 and 3.5, and the presence of heme groups in the active enzymes. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. cDNAs of 1,604 to 1,923 bp, containing open reading frames (ORF) of 508 to 513 amino acids, respectively, were cloned from a cDNA library of M. scorodonius. These data suggest glycosylation degrees of ~23% for MsP1 and 8% for MsP2. Databank homology searches revealed sequence homologies of MsP1 and MsP2 to unusual peroxidases of the fungi Thanatephorus cucumeris (DyP) and Termitomyces albuminosus (TAP).oai:repository.helmholtz-hzi.de:10033/229722019-08-30T11:30:58Zcom_10033_6832col_10033_6833
Büttner, Carina R
Sorg, Isabel
Cornelis, Guy R
Heinz, Dirk W
Niemann, Hartmut H
Division of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.
2008-04-11T08:00:00Z
2008-04-11T08:00:00Z
2008-01-25
Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD. 2008, 375 (4):997-1012 J. Mol. Biol.
1089-8638
18054956
10.1016/j.jmb.2007.11.009
http://hdl.handle.net/10033/22972
Journal of molecular biology
Many Gram-negative bacteria use a type III secretion (T3S) system to directly inject effector molecules into eucaryotic cells in order to establish a symbiotic or pathogenic relationship with their host. The translocation of many T3S proteins requires specialized chaperones from the bacterial cytosol. SycD belongs to a class of T3S chaperones that assists the secretion of pore-forming translocators and, specifically chaperones the translocators YopB and YopD from enteropathogenic Yersinia enterocolitica. In addition, SycD is involved in the regulation of virulence factor biosynthesis and secretion. In this study, we present two crystal structures of Y. enterocolitica SycD at 1.95 and 2.6 A resolution, the first experimental structures of a T3S class II chaperone specific for translocators. The fold of SycD is entirely alpha-helical and reveals three tetratricopeptide repeat-like motifs that had been predicted from amino acid sequence. In both structures, SycD forms dimers utilizing residues from the first tetratricopeptide repeat motif. Using site-directed mutagenesis and size exclusion chromatography, we verified that SycD forms head-to-head homodimers in solution. Although in both structures, dimerization largely depends on the same residues, the two assemblies represent alternative dimers that exhibit different monomer orientations and overall shape. In these two distinct head-to-head dimers, both the concave and the convex surface of each monomer are accessible for interactions with the SycD binding partners YopB and YopD. A SycD variant carrying two point mutations in the dimerization interface is properly folded but defective in dimerization. Expression of this stable SycD monomer in Yersinia does not rescue the phenotype of a sycD null mutant, suggesting a physiological relevance of the dimerization interface.
en
Amino Acid Motifs
Amino Acid Sequence
Bacterial Proteins
Biological Transport
Crystallography, X-Ray
Dimerization
Hydrogen Bonding
Hydrolysis
Hydrophobicity
Models, Molecular
Molecular Chaperones
Molecular Sequence Data
Molecular Weight
Point Mutation
Protein Structure, Quaternary
Protein Structure, Secondary
Sequence Homology, Amino Acid
Trypsin
Virulence Factors
X-Ray Diffraction
Yersinia enterocolitica
Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD.
Article2018-06-13T00:56:12ZMany Gram-negative bacteria use a type III secretion (T3S) system to directly inject effector molecules into eucaryotic cells in order to establish a symbiotic or pathogenic relationship with their host. The translocation of many T3S proteins requires specialized chaperones from the bacterial cytosol. SycD belongs to a class of T3S chaperones that assists the secretion of pore-forming translocators and, specifically chaperones the translocators YopB and YopD from enteropathogenic Yersinia enterocolitica. In addition, SycD is involved in the regulation of virulence factor biosynthesis and secretion. In this study, we present two crystal structures of Y. enterocolitica SycD at 1.95 and 2.6 A resolution, the first experimental structures of a T3S class II chaperone specific for translocators. The fold of SycD is entirely alpha-helical and reveals three tetratricopeptide repeat-like motifs that had been predicted from amino acid sequence. In both structures, SycD forms dimers utilizing residues from the first tetratricopeptide repeat motif. Using site-directed mutagenesis and size exclusion chromatography, we verified that SycD forms head-to-head homodimers in solution. Although in both structures, dimerization largely depends on the same residues, the two assemblies represent alternative dimers that exhibit different monomer orientations and overall shape. In these two distinct head-to-head dimers, both the concave and the convex surface of each monomer are accessible for interactions with the SycD binding partners YopB and YopD. A SycD variant carrying two point mutations in the dimerization interface is properly folded but defective in dimerization. Expression of this stable SycD monomer in Yersinia does not rescue the phenotype of a sycD null mutant, suggesting a physiological relevance of the dimerization interface.oai:repository.helmholtz-hzi.de:10033/234932019-08-30T11:26:07Zcom_10033_6832col_10033_6833
Wendt, K Ulrich
Weiss, Manfred S
Cramer, Patrick
Heinz, Dirk W
Department of Chemical and Analytical Sciences at Sanofi-Aventis, D-65926 Frankfurt, Germany.
2008-04-15T14:06:28Z
2008-04-15T14:06:28Z
2008-02
Structures and diseases. 2008, 15 (2):117-20 Nat. Struct. Mol. Biol.
1545-9985
18250627
10.1038/nsmb0208-117
http://hdl.handle.net/10033/23493
Nature structural & molecular biology
en
Bacterial Proteins
Protein Folding
Proteins
Protozoan Proteins
Viral Proteins
Structures and diseases.
Article2018-06-13T00:15:24Zoai:repository.helmholtz-hzi.de:10033/239922019-08-30T11:28:24Zcom_10033_6832col_10033_6833
Niemann, Hartmut H
Petoukhov, Maxim V
Härtlein, Michael
Moulin, Martine
Gherardi, Ermanno
Timmins, Peter
Heinz, Dirk W
Svergun, Dmitri I
Division of Structural Biology, Helmholtz Center for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.
2008-04-22T11:00:11Z
2008-04-22T11:00:11Z
2008-03-21
X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB. 2008, 377 (2):489-500 J. Mol. Biol.
1089-8638
18262542
10.1016/j.jmb.2008.01.027
http://hdl.handle.net/10033/23992
Journal of molecular biology
The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB(321)) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB(321) consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB(321) in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB(321) binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB(321). These results call into question whether receptor dimerization is the basic underlying event in InlB(321)-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB(321) bind and activate the Met receptor.
en
Animals
Bacterial Proteins
Binding Sites
CHO Cells
Cricetinae
Cricetulus
Listeria monocytogenes
Membrane Proteins
Models, Molecular
Neutrons
Protein Binding
Protein Structure, Quaternary
Repressor Proteins
Scattering, Small Angle
Solutions
X-Rays
X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB.
Article2018-06-13T04:15:23ZThe Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB(321)) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB(321) consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB(321) in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB(321) binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB(321). These results call into question whether receptor dimerization is the basic underlying event in InlB(321)-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB(321) bind and activate the Met receptor.oai:repository.helmholtz-hzi.de:10033/280522019-08-30T11:26:42Zcom_10033_6832col_10033_6833
Lüer, Corinna
Schauer, Stefan
Virus, Simone
Schubert, Wolf-Dieter
Heinz, Dirk W
Moser, Jürgen
Jahn, Dieter
Institute of Microbiology, Technical University Braunschweig, Germany.
2008-05-26T10:42:47Z
2008-05-26T10:42:47Z
2007-09
Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase. 2007, 274 (17):4609-14 FEBS J.
1742-464X
17697121
10.1111/j.1742-4658.2007.05989.x
http://hdl.handle.net/10033/28052
The FEBS journal
The initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substitution of amino acid residues involved in glutamate side chain binding (S109A, T49V, R52K) or in stabilizing the arginine 52 glutamate interaction (glutamate 54 and histidine 99) did not abrogate enzyme activity. Replacing glutamine 116 and glutamate 114, involved in glutamate-enzyme interaction near the aminoacyl bond to tRNA(Glu), by leucine and lysine, respectively, however, did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNA(Glu) represents the crucial determinant for substrate recognition by GluTR, whereas the necessity for product release by a 'back door' exit allows for a degree of structural variability in the recognition of the amino acid moiety. Analyzing the esterase activity, which occured in the absence of NADPH, of GluTR variants using the substrate 4-nitrophenyl acetate confirmed the crucial role of cysteine 50 for thioester formation. Finally, the GluTR variant Q116L was observed to lack reductase activity whereas esterase activity was retained. Structure-based molecular modeling indicated that glutamine 116 may be crucial in positioning the nicotinamide group of NADPH to allow for productive hydride transfer to the substrate. Our data thus provide new information about the distinct function of active site residues of GluTR from E. coli.
en
Aldehyde Oxidoreductases
Base Sequence
Catalysis
Chromatography, High Pressure Liquid
DNA Primers
Escherichia coli
Glutamic Acid
Hydrogen
Kinetics
Mutagenesis, Site-Directed
Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase.
Article2008-09-05T00:00:00ZThe initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substitution of amino acid residues involved in glutamate side chain binding (S109A, T49V, R52K) or in stabilizing the arginine 52 glutamate interaction (glutamate 54 and histidine 99) did not abrogate enzyme activity. Replacing glutamine 116 and glutamate 114, involved in glutamate-enzyme interaction near the aminoacyl bond to tRNA(Glu), by leucine and lysine, respectively, however, did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNA(Glu) represents the crucial determinant for substrate recognition by GluTR, whereas the necessity for product release by a 'back door' exit allows for a degree of structural variability in the recognition of the amino acid moiety. Analyzing the esterase activity, which occured in the absence of NADPH, of GluTR variants using the substrate 4-nitrophenyl acetate confirmed the crucial role of cysteine 50 for thioester formation. Finally, the GluTR variant Q116L was observed to lack reductase activity whereas esterase activity was retained. Structure-based molecular modeling indicated that glutamine 116 may be crucial in positioning the nicotinamide group of NADPH to allow for productive hydride transfer to the substrate. Our data thus provide new information about the distinct function of active site residues of GluTR from E. coli.oai:repository.helmholtz-hzi.de:10033/303232019-08-30T11:29:47Zcom_10033_6832col_10033_6833
Schultheis, Ellen
Dreger, Michael A
Nimtz, Manfred
Wray, Victor
Hempel, Dietmar C
Nörtemann, Bernd
Institute of Biochemical Engineering, Technical University of Braunschweig, Braunschweig, Germany.
2008-06-23T13:38:12Z
2008-06-23T13:38:12Z
2008-04
Structural characterization of the exopolysaccharide PS-EDIV from Sphingomonas pituitosa strain DSM 13101. 2008, 78 (6):1017-24 Appl. Microbiol. Biotechnol.
0175-7598
18286278
10.1007/s00253-008-1383-8
http://hdl.handle.net/10033/30323
Applied microbiology and biotechnology
Members of the bacterial genus Sphingomonas are known to produce highly viscous polysaccharides in solution. The exopolysaccharide PS-EDIV was produced by Sphingomonas pituitosa strain DSM 13101, purified using centrifugation, and precipitation and its structure was elucidated by 1D and 2D NMR techniques and chemical microderivatization combined with various mass spectrometric techniques. The following repeating unit of the polysaccharide could be identified: [formula: see text]. In addition, the polysaccharide also contains acetyl and glyceryl groups whose exact positions were not determined. PS-EDIV is similar in structure to a known exopolysaccharide but differs in being the first bacterial polysaccharide in which two different glucuronic acids are combined. It caused a high viscosity of the culture broth after cultivation for 48 h, although a gelation was not observed.
en
Structural characterization of the exopolysaccharide PS-EDIV from Sphingomonas pituitosa strain DSM 13101.
Article2018-06-13T04:09:50ZMembers of the bacterial genus Sphingomonas are known to produce highly viscous polysaccharides in solution. The exopolysaccharide PS-EDIV was produced by Sphingomonas pituitosa strain DSM 13101, purified using centrifugation, and precipitation and its structure was elucidated by 1D and 2D NMR techniques and chemical microderivatization combined with various mass spectrometric techniques. The following repeating unit of the polysaccharide could be identified: [formula: see text]. In addition, the polysaccharide also contains acetyl and glyceryl groups whose exact positions were not determined. PS-EDIV is similar in structure to a known exopolysaccharide but differs in being the first bacterial polysaccharide in which two different glucuronic acids are combined. It caused a high viscosity of the culture broth after cultivation for 48 h, although a gelation was not observed.oai:repository.helmholtz-hzi.de:10033/361322019-08-30T11:33:05Zcom_10033_6832col_10033_6833
Ganzlin, Markus
Rinas, Ursula
Helmholtz Centre for Infection Research, (Former German Research Centre for Biotechnology, GBF), Inhoffenstrasse 7, Braunschweig, Germany.
2008-08-21T11:44:25Z
2008-08-21T11:44:25Z
2008-06-30
In-depth analysis of the Aspergillus niger glucoamylase (glaA) promoter performance using high-throughput screening and controlled bioreactor cultivation techniques. 2008, 135 (3):266-71 J. Biotechnol.
0168-1656
18501461
10.1016/j.jbiotec.2008.04.005
http://hdl.handle.net/10033/36132
Journal of biotechnology
An in-depth characterization of the Aspergillus niger glucoamylase (glaA) promoter performance was carried out on defined medium employing multi-well high-throughput screening as well as controlled batch and fed-batch bioreactor culture techniques with GFP as a fluorescent reporter protein. A variety of metabolizable carbon substrates and non-metabolizable analogs were screened with regard to their effect on the glaA expression system. The results clearly demonstrate that only starch and its hydrolytic products, including glucose, act as inducers. However, induction of the glaA expression system through the monosaccharide glucose is significantly lower compared to starch and the higher molecular weight starch degradation products. All other 26 carbon substrates tested do not induce, or even, as in the case of the easily metabolizable monosaccharide xylose, repress glaA-promoter controlled gene expression in the presence of the inducing disaccharide maltose with an increase of repression strength by increasing xylose concentrations. The complex effect of glucose on glaA-promoter controlled expression was also analyzed using non-metabolizable glucose analogs, namely 5-thio-glucose and 2-deoxyglucose, which were identified as novel and potent inducers of the glaA expression system. The results show that the induction strength depends on the inducer concentration with a maximum at defined concentrations and lower induction or even repression at concentrations above. Moreover, controlled fed-batch cultivations using a high maltose feed rate with concomitant extracellular accumulation of glucose resulted in lower levels of the reporter protein compared to cultures with a low-maltose feed rate without extracellular glucose accumulation, thus supporting the conclusion that increasing the glucose concentration beyond a critical point reduces the induction strength or may even cause repression. This way, the speed of polymer hydrolysis, glucose uptake and intracellular breakdown can be fine-tuned for optimal fungal growth and the metabolic burden for glucoamylase synthesis can be limited adequately in response to nutrient availability.
en
In-depth analysis of the Aspergillus niger glucoamylase (glaA) promoter performance using high-throughput screening and controlled bioreactor cultivation techniques.
Article2018-06-12T22:58:27ZAn in-depth characterization of the Aspergillus niger glucoamylase (glaA) promoter performance was carried out on defined medium employing multi-well high-throughput screening as well as controlled batch and fed-batch bioreactor culture techniques with GFP as a fluorescent reporter protein. A variety of metabolizable carbon substrates and non-metabolizable analogs were screened with regard to their effect on the glaA expression system. The results clearly demonstrate that only starch and its hydrolytic products, including glucose, act as inducers. However, induction of the glaA expression system through the monosaccharide glucose is significantly lower compared to starch and the higher molecular weight starch degradation products. All other 26 carbon substrates tested do not induce, or even, as in the case of the easily metabolizable monosaccharide xylose, repress glaA-promoter controlled gene expression in the presence of the inducing disaccharide maltose with an increase of repression strength by increasing xylose concentrations. The complex effect of glucose on glaA-promoter controlled expression was also analyzed using non-metabolizable glucose analogs, namely 5-thio-glucose and 2-deoxyglucose, which were identified as novel and potent inducers of the glaA expression system. The results show that the induction strength depends on the inducer concentration with a maximum at defined concentrations and lower induction or even repression at concentrations above. Moreover, controlled fed-batch cultivations using a high maltose feed rate with concomitant extracellular accumulation of glucose resulted in lower levels of the reporter protein compared to cultures with a low-maltose feed rate without extracellular glucose accumulation, thus supporting the conclusion that increasing the glucose concentration beyond a critical point reduces the induction strength or may even cause repression. This way, the speed of polymer hydrolysis, glucose uptake and intracellular breakdown can be fine-tuned for optimal fungal growth and the metabolic burden for glucoamylase synthesis can be limited adequately in response to nutrient availability.oai:repository.helmholtz-hzi.de:10033/477142019-08-30T11:32:16Zcom_10033_6832col_10033_6833
Gasser, Brigitte
Saloheimo, Markku
Rinas, Ursula
Dragosits, Martin
Rodríguez-Carmona, Escarlata
Baumann, Kristin
Giuliani, Maria
Parrilli, Ermenegilda
Branduardi, Paola
Lang, Christine
Porro, Danilo
Ferrer, Pau
Tutino, Maria Luisa
Mattanovich, Diethard
Villaverde, Antonio
University of Natural Resources and Applied Life Sciences Vienna, Department of Biotechnology, Vienna, Austria. diethard.mattanovich@boku.ac.at.
2009-01-19T15:22:12Z
2009-01-19T15:22:12Z
2008
Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. 2008, 7:11 Microb. Cell Fact.
1475-2859
18394160
10.1186/1475-2859-7-11
http://hdl.handle.net/10033/47714
Microbial cell factories
ABSTRACT: Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.
en
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18394160
Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview.
Article2018-06-13T03:57:14ZABSTRACT: Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.oai:repository.helmholtz-hzi.de:10033/481582019-08-30T11:30:32Zcom_10033_48156com_10033_6832col_10033_48157
Puchałka, Jacek
Oberhardt, Matthew A
Godinho, Miguel
Bielecka, Agata
Regenhardt, Daniela
Timmis, Kenneth N
Papin, Jason A
Martins dos Santos, Vítor A P
Synthetic and Systems Biology Group, Helmholtz Center for Infection Research (HZI), Braunschweig, Germany.
2009-01-28T13:53:44Z
2009-01-28T13:53:44Z
2008-10
Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology. 2008, 4 (10):e1000210 PLoS Comput. Biol.
1553-7358
18974823
10.1371/journal.pcbi.1000210
http://hdl.handle.net/10033/48158
PLoS computational biology
A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA) enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, (13)C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype-phenotype relationships and provides a sound framework to explore this versatile bacterium and to capitalize on its vast biotechnological potential.
en
http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000210
Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology.
Article2018-06-13T04:19:43ZA cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA) enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, (13)C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype-phenotype relationships and provides a sound framework to explore this versatile bacterium and to capitalize on its vast biotechnological potential.oai:repository.helmholtz-hzi.de:10033/483162019-08-30T11:26:41Zcom_10033_6832col_10033_6833
Nath, Sunil
Structural Biology, Helmholtz Centre for Infection Research
2009-02-02T10:28:41Z
2009-02-02T10:28:41Z
2008-09
14220067
10.3390/ijms9091784
http://hdl.handle.net/10033/48316
International Journal of Molecular Sciences
Molecular Diversity Preservation International
The new Unified Theory of ATP Synthesis/Hydrolysis and Muscle Contravtion, Its Manifold Fundamental Consequences and Mechanistic Implications and Its Applications in Health and Disease
Article2018-06-13T05:30:42Zoai:repository.helmholtz-hzi.de:10033/483212019-08-30T11:33:30Zcom_10033_6832col_10033_6833
Wiesand, Ulrich
Sorg, Isabel
Amstutz, Marlise
Wagner, Stefanie
van den Heuvel, Joop
Lührs, Thorsten
Cornelis, Guy R
Heinz, Dirk W
Division of Structural Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany.
2009-02-02T14:54:39Z
2009-02-02T14:54:39Z
2009-01-23
Structure of the type III secretion recognition protein YscU from Yersinia enterocolitica. 2009, 385 (3):854-66 J. Mol. Biol.
1089-8638
18976663
10.1016/j.jmb.2008.10.034
http://hdl.handle.net/10033/48321
Journal of molecular biology
The inner-membrane protein YscU has an important role during the assembly of the Yersinia enterocolitica type III secretion injectisome. Its cytoplasmic domain (YscU(C)) recognizes translocators as individual substrates in the export hierarchy. Activation of YscU entails autocleavage at a conserved NPTH motif. Modification of this motif markedly changes the properties of YscU, including translocator export cessation and production of longer injectisome needles. We determined the crystal structures of the uncleaved variants N263A and N263D of YscU(C) at 2.05 A and 1.55 A resolution, respectively. The globular domain is found to consist of a central, mixed beta-sheet surrounded by alpha-helices. The NPTH motif forms a type II beta-turn connecting two beta-strands. NMR analysis of cleaved and uncleaved YscU(C) indicates that the global structure of the protein is retained in cleaved YscU(C). The structure of YscU(C) variant N263D reveals that wild type YscU(C) is poised for cleavage due to an optimal reaction geometry for nucleophilic attack of the scissile bond by the side chain of Asn263. In vivo analysis of N263Q and H266A/R314A YscU variants showed a phenotype that combines the absence of translocator secretion with normal needle-length control. Comparing the structure of YscU to those of related proteins reveals that the linker domain between the N-terminal transmembrane domain and the autocleavage domain can switch from an extended to a largely alpha-helical conformation, allowing for optimal positioning of the autocleavage domain during injectisome assembly.
en
Structure of the type III secretion recognition protein YscU from Yersinia enterocolitica.
Article2018-06-13T04:24:06ZThe inner-membrane protein YscU has an important role during the assembly of the Yersinia enterocolitica type III secretion injectisome. Its cytoplasmic domain (YscU(C)) recognizes translocators as individual substrates in the export hierarchy. Activation of YscU entails autocleavage at a conserved NPTH motif. Modification of this motif markedly changes the properties of YscU, including translocator export cessation and production of longer injectisome needles. We determined the crystal structures of the uncleaved variants N263A and N263D of YscU(C) at 2.05 A and 1.55 A resolution, respectively. The globular domain is found to consist of a central, mixed beta-sheet surrounded by alpha-helices. The NPTH motif forms a type II beta-turn connecting two beta-strands. NMR analysis of cleaved and uncleaved YscU(C) indicates that the global structure of the protein is retained in cleaved YscU(C). The structure of YscU(C) variant N263D reveals that wild type YscU(C) is poised for cleavage due to an optimal reaction geometry for nucleophilic attack of the scissile bond by the side chain of Asn263. In vivo analysis of N263Q and H266A/R314A YscU variants showed a phenotype that combines the absence of translocator secretion with normal needle-length control. Comparing the structure of YscU to those of related proteins reveals that the linker domain between the N-terminal transmembrane domain and the autocleavage domain can switch from an extended to a largely alpha-helical conformation, allowing for optimal positioning of the autocleavage domain during injectisome assembly.oai:repository.helmholtz-hzi.de:10033/483352019-08-30T11:24:31Zcom_10033_48156com_10033_6832col_10033_48157
Oberhardt, Matthew A
Puchałka, Jacek
Fryer, Kimberly E
Martins dos Santos, Vítor A P
Papin, Jason A
Department of Biomedical Engineering, University of Virginia Health System, Box 800759, Charlottesville, VA 22908, USA.
2009-02-02T15:11:37Z
2009-02-02T15:11:37Z
2008-04
Genome-scale metabolic network analysis of the opportunistic pathogen Pseudomonas aeruginosa PAO1. 2008, 190 (8):2790-803 J. Bacteriol.
1098-5530
18192387
10.1128/JB.01583-07
http://hdl.handle.net/10033/48335
Journal of bacteriology
Pseudomonas aeruginosa is a major life-threatening opportunistic pathogen that commonly infects immunocompromised patients. This bacterium owes its success as a pathogen largely to its metabolic versatility and flexibility. A thorough understanding of P. aeruginosa's metabolism is thus pivotal for the design of effective intervention strategies. Here we aim to provide, through systems analysis, a basis for the characterization of the genome-scale properties of this pathogen's versatile metabolic network. To this end, we reconstructed a genome-scale metabolic network of Pseudomonas aeruginosa PAO1. This reconstruction accounts for 1,056 genes (19% of the genome), 1,030 proteins, and 883 reactions. Flux balance analysis was used to identify key features of P. aeruginosa metabolism, such as growth yield, under defined conditions and with defined knowledge gaps within the network. BIOLOG substrate oxidation data were used in model expansion, and a genome-scale transposon knockout set was compared against in silico knockout predictions to validate the model. Ultimately, this genome-scale model provides a basic modeling framework with which to explore the metabolism of P. aeruginosa in the context of its environmental and genetic constraints, thereby contributing to a more thorough understanding of the genotype-phenotype relationships in this resourceful and dangerous pathogen.
en
Bacterial Proteins
Computational Biology
Computer Simulation
Genes, Bacterial
Genome, Bacterial
Humans
Metabolic Networks and Pathways
Pseudomonas aeruginosa
Genome-scale metabolic network analysis of the opportunistic pathogen Pseudomonas aeruginosa PAO1.
Article2018-06-12T18:06:24ZPseudomonas aeruginosa is a major life-threatening opportunistic pathogen that commonly infects immunocompromised patients. This bacterium owes its success as a pathogen largely to its metabolic versatility and flexibility. A thorough understanding of P. aeruginosa's metabolism is thus pivotal for the design of effective intervention strategies. Here we aim to provide, through systems analysis, a basis for the characterization of the genome-scale properties of this pathogen's versatile metabolic network. To this end, we reconstructed a genome-scale metabolic network of Pseudomonas aeruginosa PAO1. This reconstruction accounts for 1,056 genes (19% of the genome), 1,030 proteins, and 883 reactions. Flux balance analysis was used to identify key features of P. aeruginosa metabolism, such as growth yield, under defined conditions and with defined knowledge gaps within the network. BIOLOG substrate oxidation data were used in model expansion, and a genome-scale transposon knockout set was compared against in silico knockout predictions to validate the model. Ultimately, this genome-scale model provides a basic modeling framework with which to explore the metabolism of P. aeruginosa in the context of its environmental and genetic constraints, thereby contributing to a more thorough understanding of the genotype-phenotype relationships in this resourceful and dangerous pathogen.oai:repository.helmholtz-hzi.de:10033/519732019-08-30T11:33:30Zcom_10033_6832col_10033_6833
Niemann, Hartmut H
Jäger, Volker
Butler, P Jonathan G
van den Heuvel, Joop
Schmidt, Sabine
Ferraris, Davide
Gherardi, Ermanno
Heinz, Dirk W
Division of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.
2009-03-04T10:23:57Z
2009-03-04T10:23:57Z
2007-07-27
Structure of the human receptor tyrosine kinase met in complex with the Listeria invasion protein InlB. 2007, 130 (2):235-46 Cell
0092-8674
17662939
10.1016/j.cell.2007.05.037
http://hdl.handle.net/10033/51973
Cell
The tyrosine kinase Met, the product of the c-met proto-oncogene and the receptor for hepatocyte growth factor/scatter factor (HGF/SF), mediates signals critical for cell survival and migration. The human pathogen Listeria monocytogenes exploits Met signaling for invasion of host cells via its surface protein InlB. We present the crystal structure of the complex between a large fragment of the human Met ectodomain and the Met-binding domain of InlB. The concave face of the InlB leucine-rich repeat region interacts tightly with the first immunoglobulin-like domain of the Met stalk, a domain which does not bind HGF/SF. A second contact between InlB and the Met Sema domain locks the otherwise flexible receptor in a rigid, signaling competent conformation. Full Met activation requires the additional C-terminal domains of InlB which induce heparin-mediated receptor clustering and potent signaling. Thus, although it elicits a similar cellular response, InlB is not a structural mimic of HGF/SF.
en
Bacterial Proteins
Binding Sites
Heparin
Hepatocyte Growth Factor
Humans
Listeria monocytogenes
Membrane Proteins
Models, Biological
Models, Molecular
Protein Binding
Protein Interaction Mapping
Protein Structure, Secondary
Protein Structure, Tertiary
Proteins
Proto-Oncogene Proteins c-met
Structure of the human receptor tyrosine kinase met in complex with the Listeria invasion protein InlB.
Article2018-06-13T07:43:51ZThe tyrosine kinase Met, the product of the c-met proto-oncogene and the receptor for hepatocyte growth factor/scatter factor (HGF/SF), mediates signals critical for cell survival and migration. The human pathogen Listeria monocytogenes exploits Met signaling for invasion of host cells via its surface protein InlB. We present the crystal structure of the complex between a large fragment of the human Met ectodomain and the Met-binding domain of InlB. The concave face of the InlB leucine-rich repeat region interacts tightly with the first immunoglobulin-like domain of the Met stalk, a domain which does not bind HGF/SF. A second contact between InlB and the Met Sema domain locks the otherwise flexible receptor in a rigid, signaling competent conformation. Full Met activation requires the additional C-terminal domains of InlB which induce heparin-mediated receptor clustering and potent signaling. Thus, although it elicits a similar cellular response, InlB is not a structural mimic of HGF/SF.oai:repository.helmholtz-hzi.de:10033/650552019-08-30T11:33:05Zcom_10033_6832col_10033_6833
Gurramkonda, Chandrasekhar
Adnan, Ahmad
Gäbel, Thomas
Lünsdorf, Heinrich
Ross, Anton
Nemani, Satish Kumar
Swaminathan, Sathyamangalam
Khanna, Navin
Rinas, Ursula
Helmholtz Centre for Infection Research, Braunschweig, Germany. ursula.rinas@helmholtz-hzi.de.
2009-04-16T10:59:53Z
2009-04-16T10:59:53Z
2009
Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen. 2009, 8:13 Microb. Cell Fact.
1475-2859
19208244
10.1186/1475-2859-8-13
http://hdl.handle.net/10033/65055
Microbial cell factories
ABSTRACT: BACKGROUND: Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. RESULTS: Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth. CONCLUSION: In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.
en
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=19208244
Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen.
Article2018-06-13T21:34:12ZABSTRACT: BACKGROUND: Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. RESULTS: Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth. CONCLUSION: In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.oai:repository.helmholtz-hzi.de:10033/712332019-08-30T11:34:48Zcom_10033_6832col_10033_6833
de Beer, Dalene
Jerz, Gerold
Joubert, Elizabeth
Wray, Victor
Winterhalter, Peter
ARC Infruitec-Nietvoorbij, Stellenbosch, South Africa. dbeerd@arc.agric.za
2009-06-23T08:26:46Z
2009-06-23T08:26:46Z
2009-05-08
Isolation of isomangiferin from honeybush (Cyclopia subternata) using high-speed counter-current chromatography and high-performance liquid chromatography. 2009, 1216 (19):4282-9 J Chromatogr A
1873-3778
19272608
10.1016/j.chroma.2009.02.056
http://hdl.handle.net/10033/71233
Journal of chromatography. A
Isomangiferin was isolated from Cyclopia subternata using a multi-step process including extraction, liquid-liquid partitioning, high-speed counter-current chromatography (HSCCC) and semi-preparative reversed-phase high-performance liquid chromatography (HPLC). Enrichment of phenolic compounds in a methanol extract of C. subternata leaves was conducted using liquid-liquid partitioning with ethyl acetate-methanol-water (1:1:2, v/v). The enriched fraction was further fractionated using HSCCC with a ternary solvent system consisting of tert-butyl methyl ether-n-butanol-acetonitrile-water (3:1:1:5, v/v). Isomangiferin was isolated by semi-preparative reversed-phase HPLC from a fraction containing mostly mangiferin and isomangiferin. The chemical structure of isomangiferin was confirmed by LC-high-resolution electrospray ionization MS, as well as one- and two-dimensional NMR spectroscopy.
en
Isolation of isomangiferin from honeybush (Cyclopia subternata) using high-speed counter-current chromatography and high-performance liquid chromatography.
Article2018-06-12T23:51:00ZIsomangiferin was isolated from Cyclopia subternata using a multi-step process including extraction, liquid-liquid partitioning, high-speed counter-current chromatography (HSCCC) and semi-preparative reversed-phase high-performance liquid chromatography (HPLC). Enrichment of phenolic compounds in a methanol extract of C. subternata leaves was conducted using liquid-liquid partitioning with ethyl acetate-methanol-water (1:1:2, v/v). The enriched fraction was further fractionated using HSCCC with a ternary solvent system consisting of tert-butyl methyl ether-n-butanol-acetonitrile-water (3:1:1:5, v/v). Isomangiferin was isolated by semi-preparative reversed-phase HPLC from a fraction containing mostly mangiferin and isomangiferin. The chemical structure of isomangiferin was confirmed by LC-high-resolution electrospray ionization MS, as well as one- and two-dimensional NMR spectroscopy.oai:repository.helmholtz-hzi.de:10033/864412019-08-30T11:34:48Zcom_10033_6832col_10033_6833
Kayser, Hartmut
Mann, Karlheinz
Machaidze, Gia
Nimtz, Manfred
Ringler, Philippe
Müller, Shirley A
Aebi, Ueli
Institut für Allgemeine Zoologie und Endokrinologie, Universität Ulm, Germany. hartmut.kayser@uni-ulm.de
2009-11-19T10:08:02Z
2009-11-19T10:08:02Z
2009-05-29
Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family. 2009, 389 (1):74-89 J. Mol. Biol.
1089-8638
19361516
10.1016/j.jmb.2009.03.075
http://hdl.handle.net/10033/86441
Journal of molecular biology
The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI approximately 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.
en
Amino Acid Sequence
Animals
Carbohydrate Conformation
Chromatography, Gel
Electrophoresis, Polyacrylamide Gel
Glycosylation
Imaging, Three-Dimensional
Insect Proteins
Ligands
Models, Molecular
Molecular Sequence Data
Molecular Weight
Moths
Peptides
Protein Structure, Quaternary
Sequence Homology, Amino Acid
Spectrometry, Mass, Electrospray Ionization
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Spectrophotometry, Ultraviolet
Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family.
Article2018-06-13T00:37:13ZThe abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI approximately 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.oai:repository.helmholtz-hzi.de:10033/929012019-08-30T11:34:48Zcom_10033_6832col_10033_6833
Ferraris, Davide M
Gherardi, Ermanno
Di, Ying
Heinz, Dirk W
Niemann, Hartmut H
Division of Structural Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany. yyoshiki@riken.jp
2010-02-24T13:04:40Z
2010-02-24T13:04:40Z
2010-01-22
Ligand-mediated dimerization of the Met Receptor tyrosine kinase by the bacterial invasion protein InlB. 2010, 395 (3):522-32 J. Mol. Biol.
1089-8638
19900460
10.1016/j.jmb.2009.10.074
http://hdl.handle.net/10033/92901
Journal of molecular biology
The Listeria monocytogenes surface protein InlB mediates bacterial invasion into host cells by activating the human receptor tyrosine kinase Met. So far, it is unknown how InlB or the physiological Met ligand hepatocyte growth factor/scatter factor causes Met dimerization, which is considered a prerequisite for receptor activation. We determined two new structures of InlB, revealing a recurring, antiparallel, dimeric arrangement, in which the two protomers interact through the convex face of the leucine-rich repeat domain. The same contact is found in one structure of the InlB-Met complex. Mutations disrupting the interprotomeric contact of InlB reduced its ability to activate Met and downstream signaling. Conversely, stabilization of this crystal contact by two intermolecular disulfide bonds generates a constitutively dimeric InlB variant with exceptionally high signaling activity, which can stimulate cell motility and cell division. These data demonstrate that the signaling-competent InlB-Met complex assembles with 2:2 stoichiometry around a back-to-back InlB dimer, enabling the direct contact between the stalk region of two Met molecules.
en
Ligand-mediated dimerization of the Met Receptor tyrosine kinase by the bacterial invasion protein InlB.
Article2018-06-13T01:16:37ZThe Listeria monocytogenes surface protein InlB mediates bacterial invasion into host cells by activating the human receptor tyrosine kinase Met. So far, it is unknown how InlB or the physiological Met ligand hepatocyte growth factor/scatter factor causes Met dimerization, which is considered a prerequisite for receptor activation. We determined two new structures of InlB, revealing a recurring, antiparallel, dimeric arrangement, in which the two protomers interact through the convex face of the leucine-rich repeat domain. The same contact is found in one structure of the InlB-Met complex. Mutations disrupting the interprotomeric contact of InlB reduced its ability to activate Met and downstream signaling. Conversely, stabilization of this crystal contact by two intermolecular disulfide bonds generates a constitutively dimeric InlB variant with exceptionally high signaling activity, which can stimulate cell motility and cell division. These data demonstrate that the signaling-competent InlB-Met complex assembles with 2:2 stoichiometry around a back-to-back InlB dimer, enabling the direct contact between the stalk region of two Met molecules.oai:repository.helmholtz-hzi.de:10033/1102362019-08-30T11:35:39Zcom_10033_6832col_10033_6833
Klink, Björn U
Barden, Stephan
Heidler, Thomas V
Borchers, Christina
Ladwein, Markus
Stradal, Theresia E B
Rottner, Klemens
Heinz, Dirk W
Division of Structural Biology, Helmholtz Zentrum für Infektionsforschung, D-38124 Braunschweig, Germany.
2010-08-24T09:31:19Z
2010-08-24T09:31:19Z
2010-05-28
Structure of Shigella IpgB2 in complex with human RhoA: implications for the mechanism of bacterial guanine nucleotide exchange factor mimicry. 2010, 285 (22):17197-208 J. Biol. Chem.
1083-351X
20363740
10.1074/jbc.M110.107953
http://hdl.handle.net/10033/110236
The Journal of biological chemistry
A common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP.Mg(2+) complex is preceded by the displacement of the metal ion to the alpha-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family.
en
Cloning, Molecular
Cytoskeleton
Guanine Nucleotide Exchange Factors
Guanosine Diphosphate
Guanosine Triphosphate
Humans
Ions
Magnesium
Metals
Nucleotides
Protein Binding
Protein Conformation
Shigella flexneri
rac1 GTP-Binding Protein
rhoA GTP-Binding Protein
Structure of Shigella IpgB2 in complex with human RhoA: implications for the mechanism of bacterial guanine nucleotide exchange factor mimicry.
Article2011-05-15T00:00:00ZA common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP.Mg(2+) complex is preceded by the displacement of the metal ion to the alpha-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family.oai:repository.helmholtz-hzi.de:10033/1114172019-08-30T11:34:48Zcom_10033_6832col_10033_6833
Klink, Björn U
Barden, Stephan
Heidler, Thomas V
Borchers, Christina
Ladwein, Markus
Stradal, Theresia E B
Rottner, Klemens
Heinz, Dirk W
Division of Structural Biology, Helmholtz Zentrum für Infektionsforschung, D-38124 Braunschweig, Germany.
2010-09-20T09:43:36Z
2010-09-20T09:43:36Z
2010-05-28
Structure of Shigella IpgB2 in complex with human RhoA: implications for the mechanism of bacterial guanine nucleotide exchange factor mimicry. 2010, 285 (22):17197-208 J. Biol. Chem.
1083-351X
20363740
10.1074/jbc.M110.107953
http://hdl.handle.net/10033/111417
The Journal of biological chemistry
A common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP.Mg(2+) complex is preceded by the displacement of the metal ion to the alpha-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family.
en
Cloning, Molecular
Cytoskeleton
Guanine Nucleotide Exchange Factors
Guanosine Diphosphate
Guanosine Triphosphate
Humans
Ions
Magnesium
Metals
Nucleotides
Protein Binding
Protein Conformation
Shigella flexneri
rac1 GTP-Binding Protein
rhoA GTP-Binding Protein
Structure of Shigella IpgB2 in complex with human RhoA: implications for the mechanism of bacterial guanine nucleotide exchange factor mimicry.
Article2011-06-15T00:00:00ZA common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP.Mg(2+) complex is preceded by the displacement of the metal ion to the alpha-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family.oai:repository.helmholtz-hzi.de:10033/1192102019-08-30T11:35:39Zcom_10033_6832col_10033_6833
Kämpfer, Peter
Busse, Hans-Jürgen
Tindall, Brian J
Nimtz, Manfred
Grün-Wollny, Iris
Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany. peter.kaempfer@umwelt.uni-giessen.de
2011-01-12T15:07:59Z
2011-01-12T15:07:59Z
2010-05
Nonomuraea rosea sp. nov. 2010, 60 (Pt 5):1118-24 Int. J. Syst. Evol. Microbiol.
1466-5026
19666797
10.1099/ijs.0.014845-0
http://hdl.handle.net/10033/119210
International journal of systematic and evolutionary microbiology
A Gram-positively staining, aerobic, non-motile actinomycete, strain GW 12687(T), that formed rose-pigmented colonies and branched substrate and aerial mycelia was studied in detail for its taxonomic position. On the basis of 16S rRNA gene sequence similarity studies, strain GW 12687(T) was grouped into the genus Nonomuraea, being most closely related to Nonomuraea dietziae (97.6 %), Nonomuraea africana (97.1 %), and Nonomuraea kuesteri (97.1 %). The 16S rRNA gene sequence similarity to other species of the genus Nonomuraea was < or =97 %. The chemotaxonomic characterization supported allocation of the strain to the genus Nonomuraea. The major menaquinone was MK-9(H(4)) with minor amounts of MK-9(H(2)), MK-9(H(6)), MK-9(H(0)) and MK-8(H(4)). The polar lipid profile contained the major compound diphosphatidylglycerol, moderate amounts of phosphatidylmonomethylethanolamine, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, hydroxy-phosphatidylmonomethylethanolamine, and an unknown aminophosphoglycolipid. Phosphatidylinositol mannosides and phosphatidylinositol were also present. The major fatty acids were iso- and anteiso- and 10-methyl-branched fatty acids. The results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain GW 12687(T) from closely related species. Thus, GW 12687(T) represents a novel species of the genus Nonomuraea, for which the name Nonomuraea rosea sp. nov. is proposed, with GW 12687(T) (=DSM 45177(T) =CCUG 56107(T)) as the type strain.
en
Actinomycetales
Bacterial Typing Techniques
DNA, Bacterial
DNA, Ribosomal
Fatty Acids
Genes, rRNA
Genotype
Lipids
Molecular Sequence Data
Phenotype
Phylogeny
RNA, Ribosomal, 16S
Sequence Analysis, DNA
Soil Microbiology
Species Specificity
Vitamin K 2
Nonomuraea rosea sp. nov.
Article2011-05-15T00:00:00ZA Gram-positively staining, aerobic, non-motile actinomycete, strain GW 12687(T), that formed rose-pigmented colonies and branched substrate and aerial mycelia was studied in detail for its taxonomic position. On the basis of 16S rRNA gene sequence similarity studies, strain GW 12687(T) was grouped into the genus Nonomuraea, being most closely related to Nonomuraea dietziae (97.6 %), Nonomuraea africana (97.1 %), and Nonomuraea kuesteri (97.1 %). The 16S rRNA gene sequence similarity to other species of the genus Nonomuraea was < or =97 %. The chemotaxonomic characterization supported allocation of the strain to the genus Nonomuraea. The major menaquinone was MK-9(H(4)) with minor amounts of MK-9(H(2)), MK-9(H(6)), MK-9(H(0)) and MK-8(H(4)). The polar lipid profile contained the major compound diphosphatidylglycerol, moderate amounts of phosphatidylmonomethylethanolamine, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, hydroxy-phosphatidylmonomethylethanolamine, and an unknown aminophosphoglycolipid. Phosphatidylinositol mannosides and phosphatidylinositol were also present. The major fatty acids were iso- and anteiso- and 10-methyl-branched fatty acids. The results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain GW 12687(T) from closely related species. Thus, GW 12687(T) represents a novel species of the genus Nonomuraea, for which the name Nonomuraea rosea sp. nov. is proposed, with GW 12687(T) (=DSM 45177(T) =CCUG 56107(T)) as the type strain.oai:repository.helmholtz-hzi.de:10033/1292952019-08-30T11:37:00Zcom_10033_6832col_10033_6833
Haffke, Matthias
Menzel, Anja
Carius, Yvonne
Jahn, Dieter
Heinz, Dirk W
Helmholtz Zentrum für Infektionsforschung, Braunschweig, Germany.
2011-05-09T14:12:58Z
2011-05-09T14:12:58Z
2010-09
Structures of the nucleotide-binding domain of the human ABCB6 transporter and its complexes with nucleotides. 2010, 66 (Pt 9):979-87 Acta Crystallogr. D Biol. Crystallogr.
1399-0047
20823549
10.1107/S0907444910028593
http://hdl.handle.net/10033/129295
Acta crystallographica. Section D, Biological crystallography
The human ATP-binding cassette (ABC) transporter ABCB6 is involved in haem-precursor transport across the mitochondrial membrane. The crystal structure of its nucleotide-binding domain (NBD) has been determined in the apo form and in complexes with ADP, with ADP and Mg(2+) and with ATP at high resolution. The overall structure is L-shaped and consists of two lobes, consistent with other reported NBD structures. Nucleotide binding is mediated by the highly conserved Tyr599 and the Walker A motif, and induces notable structural changes. Structural comparison with other structurally characterized NBDs and full-length ABC transporters gives the first insight into the possible catalytic mechanism of ABCB6 and the role of the N-terminal helix alpha(1) in full-length ABCB6.
en
ATP-Binding Cassette Transporters
Amino Acid Sequence
Animals
Crystallography, X-Ray
Humans
Models, Molecular
Molecular Sequence Data
Nucleotides
Protein Interaction Domains and Motifs
Protein Structure, Tertiary
Sequence Alignment
Structural Homology, Protein
Structures of the nucleotide-binding domain of the human ABCB6 transporter and its complexes with nucleotides.
Article2018-06-13T01:17:21ZThe human ATP-binding cassette (ABC) transporter ABCB6 is involved in haem-precursor transport across the mitochondrial membrane. The crystal structure of its nucleotide-binding domain (NBD) has been determined in the apo form and in complexes with ADP, with ADP and Mg(2+) and with ATP at high resolution. The overall structure is L-shaped and consists of two lobes, consistent with other reported NBD structures. Nucleotide binding is mediated by the highly conserved Tyr599 and the Walker A motif, and induces notable structural changes. Structural comparison with other structurally characterized NBDs and full-length ABC transporters gives the first insight into the possible catalytic mechanism of ABCB6 and the role of the N-terminal helix alpha(1) in full-length ABCB6.oai:repository.helmholtz-hzi.de:10033/1358882019-08-30T11:37:23Zcom_10033_6832col_10033_6833
Darpel, Karin E
Langner, Kathrin F A
Nimtz, Manfred
Anthony, Simon J
Brownlie, Joe
Takamatsu, Haru-Hisa
Mellor, Philip S
Mertens, Peter P C
Pirbright Laboratory, Vector-borne Disease Programme, Institute for Animal Health, Woking, United Kingdom. karin.darpel@bbsrc.ac.uk
2011-07-12T14:04:34Z
2011-07-12T14:04:34Z
2011
Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles. 2011, 6 (3):e17545 PLoS ONE
1932-6203
21423801
10.1371/journal.pone.0017545
http://hdl.handle.net/10033/135888
PloS one
Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.
en
Animals
Bluetongue
Bluetongue virus
Cell Line
Ceratopogonidae
Chymotrypsin
Electrophoresis, Polyacrylamide Gel
Insect Vectors
Molecular Weight
Protease Inhibitors
Saliva
Salivary Proteins and Peptides
Sheep
Temperature
Trypsin
Viral Proteins
Virion
Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles.
Article2018-06-13T00:24:05ZBluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.oai:repository.helmholtz-hzi.de:10033/1408692019-08-30T11:37:24Zcom_10033_6832col_10033_6833
Quade, Nick
Dieckmann, Marieke
Haffke, Matthias
Heroven, Ann Kathrin
Dersch, Petra
Heinz, Dirk W
Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany.
2011-08-26T14:30:15Z
2011-08-26T14:30:15Z
2011-02
Structure of the effector-binding domain of the LysR-type transcription factor RovM from Yersinia pseudotuberculosis. 2011, 67 (Pt 2):81-90 Acta Crystallogr. D Biol. Crystallogr.
1399-0047
21245528
10.1107/S0907444910049681
http://hdl.handle.net/10033/140869
Acta crystallographica. Section D, Biological crystallography
In enteropathogenic Yersinia, the expression of several early-phase virulence factors such as invasin is tightly regulated in response to environmental cues. The responsible regulatory network is complex, involving several regulatory RNAs and proteins such as the LysR-type transcription regulator (LTTR) RovM. In this study, the crystal structure of the effector-binding domain (EBD) of RovM, the first LTTR protein described as being involved in virulence regulation, was determined at a resolution of 2.4 Å. Size-exclusion chromatography and comparison with structures of full-length LTTRs show that RovM is most likely to adopt a tetrameric arrangement with two distant DNA-binding domains (DBDs), causing the DNA to bend around it. Additionally, a cavity was detected in RovM which could bind small inducer molecules.
en
Bacterial Proteins
Crystallography, X-Ray
Ligands
Models, Molecular
Protein Structure, Quaternary
Protein Structure, Tertiary
Transcription Factors
Yersinia pseudotuberculosis
Structure of the effector-binding domain of the LysR-type transcription factor RovM from Yersinia pseudotuberculosis.
Article2018-06-13T14:17:03ZIn enteropathogenic Yersinia, the expression of several early-phase virulence factors such as invasin is tightly regulated in response to environmental cues. The responsible regulatory network is complex, involving several regulatory RNAs and proteins such as the LysR-type transcription regulator (LTTR) RovM. In this study, the crystal structure of the effector-binding domain (EBD) of RovM, the first LTTR protein described as being involved in virulence regulation, was determined at a resolution of 2.4 Å. Size-exclusion chromatography and comparison with structures of full-length LTTRs show that RovM is most likely to adopt a tetrameric arrangement with two distant DNA-binding domains (DBDs), causing the DNA to bend around it. Additionally, a cavity was detected in RovM which could bind small inducer molecules.oai:repository.helmholtz-hzi.de:10033/2023692019-08-30T11:31:49Zcom_10033_6832col_10033_6833
Strube, Christian P
Homann, Arne
Gamer, Martin
Jahn, Dieter
Seibel, Jürgen
Heinz, Dirk W
Department of Molecular Structural Biology, Helmholtz-Centre for Infection Research, Inhoffenstrasse 7B, 38124 Braunschweig, Germany.
2012-01-11T13:03:17Z
2012-01-11T13:03:17Z
2011-05-20
Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs. 2011, 286 (20):17593-600 J. Biol. Chem.
1083-351X
21454585
10.1074/jbc.M110.203166
http://hdl.handle.net/10033/202369
The Journal of biological chemistry
Despite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, β-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (k(cat)) and substrate affinity (K(m)) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases.
en
Amino Acid Motifs
Amino Acid Substitution
Bacillus megaterium
Bacterial Proteins
Hexosyltransferases
Mutation, Missense
Polysaccharides, Bacterial
Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs.
Article2012-06-15T00:00:00ZDespite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, β-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (k(cat)) and substrate affinity (K(m)) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases.oai:repository.helmholtz-hzi.de:10033/2087492019-08-30T11:30:58Zcom_10033_6832col_10033_6833
Busso, Didier
Peleg, Yoav
Heidebrecht, Tatjana
Romier, Christophe
Jacobovitch, Yossi
Dantes, Ada
Salim, Loubna
Troesch, Edouard
Schuetz, Anja
Heinemann, Udo
Folkers, Gert E
Geerlof, Arie
Wilmanns, Matthias
Polewacz, Andrea
Quedenau, Claudia
Büssow, Konrad
Adamson, Rachel
Blagova, Elena
Walton, Julia
Cartwright, Jared L
Bird, Louise E
Owens, Raymond J
Berrow, Nick S
Wilson, Keith S
Sussman, Joel L
Perrakis, Anastassis
Celie, Patrick H N
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (Inserm), U964/Centre National deRecherche Scientifique (CNRS), UMR 7104, Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch, France.
2012-02-03T12:10:12Z
2012-02-03T12:10:12Z
2011-08
Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study. 2011, 175 (2):159-70 J. Struct. Biol.
1095-8657
21382497
10.1016/j.jsb.2011.03.004
http://hdl.handle.net/10033/208749
Journal of structural biology
Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.
en
Academies and Institutes
CCAAT-Binding Factor
Cell Cycle Proteins
Cloning, Molecular
Escherichia coli
Europe
Genetic Vectors
International Cooperation
Israel
Multiprotein Complexes
Recombinant Proteins
Transcription Factors, TFII
Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.
Article2018-06-13T19:48:24ZEscherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.oai:repository.helmholtz-hzi.de:10033/2155242019-08-30T11:25:43Zcom_10033_6832col_10033_6833
Storbeck, Sonja
Saha, Sayantan
Krausze, Joern
Klink, Björn U
Heinz, Dirk W
Layer, Gunhild
Institute of Microbiology, Technische Universität Braunschweig, 38106 Braunschweig, Germany.
2012-03-13T13:12:18Z
2012-03-13T13:12:18Z
2011-07-29
Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferases. 2011, 286 (30):26754-67 J. Biol. Chem.
1083-351X
21632530
10.1074/jbc.M111.239855
http://hdl.handle.net/10033/215524
The Journal of biological chemistry
During the biosynthesis of heme d(1), the essential cofactor of cytochrome cd(1) nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-L-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-L-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a "puckered" conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.
en
Archived with thanks to The Journal of biological chemistry
Bacterial Proteins
Catalysis
Crystallography, X-Ray
Heme
Methyltransferases
Mutagenesis, Site-Directed
Mutation, Missense
Protein Structure, Tertiary
Pseudomonas aeruginosa
Uroporphyrinogens
Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferases.
Article2012-07-15T00:00:00ZDuring the biosynthesis of heme d(1), the essential cofactor of cytochrome cd(1) nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-L-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-L-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a "puckered" conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.oai:repository.helmholtz-hzi.de:10033/2266712019-08-30T11:36:32Zcom_10033_6832col_10033_6833
Wilke, Sonja
Groebe, Lothar
Maffenbeier, Vitali
Jäger, Volker
Gossen, Manfred
Josewski, Jörn
Duda, Agathe
Polle, Lilia
Owens, Raymond J
Wirth, Dagmar
Heinz, Dirk W
van den Heuvel, Joop
Büssow, Konrad
Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
2012-05-30T08:49:37Z
2012-05-30T08:49:37Z
2011
Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. 2011, 6 (12):e27829 PLoS ONE
1932-6203
22174749
10.1371/journal.pone.0027829
http://hdl.handle.net/10033/226671
PloS one
Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).
en
Archived with thanks to PloS one
Animals
Biophysical Phenomena
CHO Cells
Cell Culture Techniques
Cell Line
Cricetinae
Cricetulus
Crystallization
DNA Nucleotidyltransferases
Genetic Vectors
Glycoproteins
Glycosylation
Green Fluorescent Proteins
Humans
Luminescent Proteins
Protein Structure, Tertiary
Recombinant Proteins
Recombination, Genetic
Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.
Article2018-06-13T19:45:48ZStudying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).oai:repository.helmholtz-hzi.de:10033/2276762019-08-30T11:36:59Zcom_10033_6832col_10033_6833
König, Sebastian
Nimtz, Manfred
Scheiter, Maxi
Ljunggren, Hans-Gustaf
Bryceson, Yenan T
Jänsch, Lothar
Department of Molecular Structural Biology, Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Germany.
2012-06-06T14:18:27Z
2012-06-06T14:18:27Z
2012
Kinome analysis of receptor-induced phosphorylation in human natural killer cells. 2012, 7 (1):e29672 PLoS ONE
1932-6203
22238634
10.1371/journal.pone.0029672
http://hdl.handle.net/10033/227676
PloS one
Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation.
en
Archived with thanks to PloS one
Amino Acid Sequence
Animals
Cells, Cultured
Cluster Analysis
Humans
K562 Cells
Killer Cells, Natural
Mice
Models, Biological
Phosphoproteins
Phosphorylation
Phosphotransferases
Phylogeny
Primary Cell Culture
Proteome
Receptors, Cell Surface
Receptors, Fc
Kinome analysis of receptor-induced phosphorylation in human natural killer cells.
Article2018-06-13T05:29:27ZNatural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation.oai:repository.helmholtz-hzi.de:10033/2309472019-08-30T11:33:28Zcom_10033_6832col_10033_6833
Langlotz, Christine
Schollmeyer, Martin
Coplin, David L.
Nimtz, Manfred
Geider, Klaus
Max-Planck-Institute for Cell Biology, Ladenburg, Germany
2012-06-27T09:29:52Z
2012-06-27T09:29:52Z
2012-06-27
Biosynthesis of the repeating units of the exopolysaccharides amylovoran from Erwinia amylovora and stewartan from Pantoea stewartii 2011, 75 (4):163 Physiological and Molecular Plant Pathology
08855765
10.1016/j.pmpp.2011.04.001
http://hdl.handle.net/10033/230947
Physiological and Molecular Plant Pathology
http://linkinghub.elsevier.com/retrieve/pii/S0885576511000269
Archived with thanks to Physiological and Molecular Plant Pathology
Biosynthesis of the repeating units of the exopolysaccharides amylovoran from Erwinia amylovora and stewartan from Pantoea stewartii
Article2018-06-13T01:17:31Zoai:repository.helmholtz-hzi.de:10033/2394712019-08-30T11:26:13Zcom_10033_6835com_10033_6832col_10033_6836
Ebrahim, Weaam
Kjer, Julia
El Amrani, Mustapha
Wray, Victor
Lin, Wenhan
Ebel, Rainer
Lai, Daowan
Proksch, Peter
Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine University, Universitaetsstrasse 1, D-40225 Duesseldorf, Germany; Email: weel-001@uni-duesseldorf.de or weaamnabil@mans.edu.eg (W.E.); jacob.julia@web.de (J.K.); mustapha.elamrani@uni-duesseldorf.de (M.E.A.).
2012-08-22T13:01:10Z
2012-08-22T13:01:10Z
2012-05
Pullularins E and F, Two New Peptides from the Endophytic Fungus Bionectria ochroleuca Isolated from the Mangrove Plant Sonneratia caseolaris. 2012, 10 (5):1081-91 Mar Drugs
1660-3397
22822358
10.3390/md10051081
http://hdl.handle.net/10033/239471
Marine drugs
Chemical investigation of the EtOAc extract of the endophytic fungus Bionectria ochroleuca, isolated from the inner leaf tissues of the plant Sonneratia caseolaris (Sonneratiaceae) from Hainan island (China), yielded two new peptides, pullularins E and F (1 and 2) together with three known compounds (3-5). The structures of the new compounds were unambiguously determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of amino acids were determined by HPLC analysis of acid hydrolysates using Marfey's method. The isolated compounds exhibited pronounced to moderate cytotoxic activity against the mouse lymphoma cells (L5178Y) with EC(50) values ranging between 0.1 and 6.7 µg/mL.
en
Archived with thanks to Marine drugs
Pullularins E and F, Two New Peptides from the Endophytic Fungus Bionectria ochroleuca Isolated from the Mangrove Plant Sonneratia caseolaris.
Article2018-06-13T15:56:03ZChemical investigation of the EtOAc extract of the endophytic fungus Bionectria ochroleuca, isolated from the inner leaf tissues of the plant Sonneratia caseolaris (Sonneratiaceae) from Hainan island (China), yielded two new peptides, pullularins E and F (1 and 2) together with three known compounds (3-5). The structures of the new compounds were unambiguously determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of amino acids were determined by HPLC analysis of acid hydrolysates using Marfey's method. The isolated compounds exhibited pronounced to moderate cytotoxic activity against the mouse lymphoma cells (L5178Y) with EC(50) values ranging between 0.1 and 6.7 µg/mL.oai:repository.helmholtz-hzi.de:10033/2451912019-08-30T11:27:46Zcom_10033_620626com_10033_6832col_10033_620627col_10033_6833
Wilke, Sonja
Krausze, Joern
Büssow, Konrad
Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstr, 7, 38124 Braunschweig, Germany. konrad@buessow.com.
2012-09-20T09:22:54Z
2012-09-20T09:22:54Z
2012
Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx. 2012, 10:62 BMC Biol.
1741-7007
22809326
10.1186/1741-7007-10-62
http://hdl.handle.net/10033/245191
BMC biology
ABSTRACT:
en
Archived with thanks to BMC biology
Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx.
Article2018-06-12T17:46:13ZABSTRACT:oai:repository.helmholtz-hzi.de:10033/2509932019-08-30T11:34:48Zcom_10033_6832col_10033_6833
de Groot, Jens C
Schlüter, Kai
Carius, Yvonne
Quedenau, Claudia
Vingadassalom, Didier
Faix, Jan
Weiss, Stefanie M
Reichelt, Joachim
Standfuss-Gabisch, Christine
Lesser, Cammie F
Leong, John M
Heinz, Dirk W
Büssow, Konrad
Stradal, Theresia E B
Division of Structural Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
2012-11-05T14:50:53Z
2012-11-05T14:50:53Z
2011-09-07
Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli. 2011, 19 (9):1294-306 Structure
1878-4186
21893288
10.1016/j.str.2011.06.015
http://hdl.handle.net/10033/250993
Structure (London, England : 1993)
Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell.
en
Archived with thanks to Structure (London, England : 1993)
Amino Acid Motifs
Amino Acid Substitution
Animals
Binding Sites
COS Cells
Calorimetry
Cercopithecus aethiops
Crystallography, X-Ray
Escherichia coli O157
Escherichia coli Proteins
Host-Pathogen Interactions
Humans
Hydrogen Bonding
Hydrophobic and Hydrophilic Interactions
Immunoprecipitation
Models, Molecular
Mutagenesis, Site-Directed
Nerve Tissue Proteins
Peptide Fragments
Protein Binding
Protein Interaction Domains and Motifs
Receptors, Cell Surface
Thermodynamics
Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli.
Article2018-06-13T05:37:29ZActin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell.oai:repository.helmholtz-hzi.de:10033/2707952019-08-30T11:27:46Zcom_10033_6832col_10033_6833
Glotzbach, Bernhard
Schmelz, Stefan
Reinwarth, Michael
Christmann, Andreas
Heinz, Dirk W
Kolmar, Harald
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Germany.
2013-03-01T09:44:17Z
2013-03-01T09:44:17Z
2013-01
Structural characterization of Spinacia oleracea trypsin inhibitor III (SOTI-III). 2013, 69 (Pt 1):114-20 Acta Crystallogr. D Biol. Crystallogr.
1399-0047
23275169
10.1107/S0907444912043880
http://hdl.handle.net/10033/270795
Acta crystallographica. Section D, Biological crystallography
In recent decades, several canonical serine protease inhibitor families have been classified and characterized. In contrast to most trypsin inhibitors, those from garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) do not share sequence similarity and have been proposed to form the new Mirabilis serine protease inhibitor family. These 30-40-amino-acid inhibitors possess a defined disulfide-bridge topology and belong to the cystine-knot miniproteins (knottins). To date, no atomic structure of this inhibitor family has been solved. Here, the first structure of S. oleracea trypsin inhibitor III (SOTI-III), in complex with bovine pancreatic trypsin, is reported. The inhibitor was synthesized by solid-phase peptide synthesis on a multi-milligram scale and was assayed to test its inhibitory activity and binding properties. The structure confirmed the proposed cystine-bridge topology. The structural features of SOTI-III suggest that it belongs to a new canonical serine protease inhibitor family with promising properties for use in protein-engineering and medical applications.
en
Archived with thanks to Acta crystallographica. Section D, Biological crystallography
Structural characterization of Spinacia oleracea trypsin inhibitor III (SOTI-III).
Article2014-01-15T00:00:00ZIn recent decades, several canonical serine protease inhibitor families have been classified and characterized. In contrast to most trypsin inhibitors, those from garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) do not share sequence similarity and have been proposed to form the new Mirabilis serine protease inhibitor family. These 30-40-amino-acid inhibitors possess a defined disulfide-bridge topology and belong to the cystine-knot miniproteins (knottins). To date, no atomic structure of this inhibitor family has been solved. Here, the first structure of S. oleracea trypsin inhibitor III (SOTI-III), in complex with bovine pancreatic trypsin, is reported. The inhibitor was synthesized by solid-phase peptide synthesis on a multi-milligram scale and was assayed to test its inhibitory activity and binding properties. The structure confirmed the proposed cystine-bridge topology. The structural features of SOTI-III suggest that it belongs to a new canonical serine protease inhibitor family with promising properties for use in protein-engineering and medical applications.oai:repository.helmholtz-hzi.de:10033/2719922019-08-30T11:26:42Zcom_10033_271853com_10033_6832col_10033_271872
Myllykoski, Matti
Raasakka, Arne
Han, Huijong
Kursula, Petri
Department of Biochemistry and Biocenter Oulu, University of Oulu, Oulu, Finland.
2013-03-13T08:07:51Z
2013-03-13T08:07:51Z
2012
Myelin 2',3'-cyclic nucleotide 3'-phosphodiesterase: active-site ligand binding and molecular conformation. 2012, 7 (2):e32336 PLoS ONE
1932-6203
22393399
10.1371/journal.pone.0032336
http://hdl.handle.net/10033/271992
PloS one
The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2'-nucleotide products from 2',3'-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2'-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain.
en
info:eu-repo/grantAgreement/EC/FP7/226716
Archived with thanks to PloS one
openAccess
2',3'-Cyclic-Nucleotide Phosphodiesterases
Animals
Catalysis
Catalytic Domain
Citric Acid
Crystallography, X-Ray
Dimerization
Ligands
Mice
Models, Molecular
Molecular Conformation
Myelin Sheath
Protein Binding
Protein Structure, Tertiary
RNA
Sulfates
Myelin 2',3'-cyclic nucleotide 3'-phosphodiesterase: active-site ligand binding and molecular conformation.
Article2018-06-13T05:24:53ZThe 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2'-nucleotide products from 2',3'-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2'-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain.oai:repository.helmholtz-hzi.de:10033/2931262019-08-30T11:34:22Zcom_10033_6832col_10033_6833
Zelena, Katerina
Takenberg, Meike
Lunkenbein, Stefan
Woche, Susanne K
Nimtz, Manfred
Berger, Ralf G
Naturwissenschaftliche Fakultät der Leibniz Universität Hannover, Institut für Lebensmittelchemie, Hannover, Germany.
2013-05-31T09:30:11Z
2013-05-31T09:30:11Z
2013-03
PfaH2: A novel hydrophobin from the ascomycete Paecilomyces farinosus. 2013, 60 (2):147-54 Biotechnol. Appl. Biochem.
1470-8744
23600571
10.1002/bab.1077
http://hdl.handle.net/10033/293126
Biotechnology and applied biochemistry
The pfah2 gene coding for a novel hydrophobin PfaH2 from the ascomycete Paecilomyces farinosus was identified during sequencing of random clones from a cDNA library. The corresponding protein sequence of PfaH2 deduced from the cDNA comprised 134 amino acids (aa). A 16 aa signal sequence preceded the N-terminus of the mature protein. PfaH2 belonged to the class Ia hydrophobins. The protein was isolated using trifluoroacetic acid extraction and purified via SDS-PAGE and high-performance liquid chromatography. The surface activity of the recently described PfaH1 and of PfaH2 was compared by the determination of contact angles (CAs) on glass slides and Teflon tape, and the CA of distilled water droplets was measured on glass slides coated with hydrophobin PfaH1 or PfaH2. Surprisingly, both hydrophobins adsorbed to hydrophilic surfaces and changed their physicochemical properties to a similar quantitative extent, although little aa sequence homology was found.
en
Archived with thanks to Biotechnology and applied biochemistry
PfaH2: A novel hydrophobin from the ascomycete Paecilomyces farinosus.
Article2018-06-13T02:26:58ZThe pfah2 gene coding for a novel hydrophobin PfaH2 from the ascomycete Paecilomyces farinosus was identified during sequencing of random clones from a cDNA library. The corresponding protein sequence of PfaH2 deduced from the cDNA comprised 134 amino acids (aa). A 16 aa signal sequence preceded the N-terminus of the mature protein. PfaH2 belonged to the class Ia hydrophobins. The protein was isolated using trifluoroacetic acid extraction and purified via SDS-PAGE and high-performance liquid chromatography. The surface activity of the recently described PfaH1 and of PfaH2 was compared by the determination of contact angles (CAs) on glass slides and Teflon tape, and the CA of distilled water droplets was measured on glass slides coated with hydrophobin PfaH1 or PfaH2. Surprisingly, both hydrophobins adsorbed to hydrophilic surfaces and changed their physicochemical properties to a similar quantitative extent, although little aa sequence homology was found.oai:repository.helmholtz-hzi.de:10033/2938552019-08-30T11:28:45Zcom_10033_6835com_10033_6832col_10033_6836
Ibrahim, Sabrin R. M.
Edrada-Ebel, RuAngelie
Mohamed,Gamal A.
Youssef, Diaa T. A
Wray, Victor
Proksch, Peter
Helmholz Centre for Infection research, D-38124 Braunschweig, Germany
2013-06-12T08:37:36Z
2013-06-12T08:37:36Z
2008-04-16
2008-01
Callyaerin G, a new cytotoxic cyclic peptide from the marine sponge Callyspongia aerizusa 2008, 2008 (12):164 Arkivoc
1551-7012
1551-7012
10.3998/ark.5550190.0009.c18
http://hdl.handle.net/10033/293855
Arkivoc
Arkat
http://hdl.handle.net/2027/spo.5550190.0009.c18
Archived with thanks to Arkivoc
Callyaerin G, a new cytotoxic cyclic peptide from the marine sponge Callyspongia aerizusa
Article2018-06-13T14:11:34Zoai:repository.helmholtz-hzi.de:10033/2972262019-08-30T11:28:46Zcom_10033_6832col_10033_6833
Kudryashev, M.
Stenta, M.
Schmelz, S.
Amstutz, M.
Wiesand, U.
Castano-Diez, D.
Degiacomi, M. T.
Munnich, S.
Bleck, C. K.
Kowal, J.
Diepold, A.
Heinz, D. W.
Dal Peraro, M.
Cornelis, G. R.
Stahlberg, H.
Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany
2013-08-01T13:33:22Z
2013-08-01T13:33:22Z
2013-07-30
In situ structural analysis of the Yersinia enterocolitica injectisome 2013, 2 (0):e00792 eLife
2050-084X
10.7554/eLife.00792
http://hdl.handle.net/10033/297226
eLife
http://elife.elifesciences.org/lookup/doi/10.7554/eLife.00792
Archived with thanks to eLife
In situ structural analysis of the Yersinia enterocolitica injectisome
Article2018-06-13T00:24:29Zoai:repository.helmholtz-hzi.de:10033/3025902019-08-30T11:33:55Zcom_10033_6835com_10033_6832col_10033_6836
Wang, Jian-ping
Lin, Wenhan
Wray, Victor
Lai, Daowan
Proksch, Peter
2013-10-01T14:53:25Z
2013-10-01T14:53:25Z
2013-10-01
Induced production of depsipeptides by co-culturing Fusarium tricinctum and Fusarium begoniae 2013, 54 (20):2492 Tetrahedron Letters
00404039
10.1016/j.tetlet.2013.03.005
http://hdl.handle.net/10033/302590
Tetrahedron Letters
http://linkinghub.elsevier.com/retrieve/pii/S0040403913003869
Archived with thanks to Tetrahedron Letters
Induced production of depsipeptides by co-culturing Fusarium tricinctum and Fusarium begoniae
Article2018-06-13T15:12:11Zoai:repository.helmholtz-hzi.de:10033/3029852019-08-30T11:37:00Zcom_10033_48156com_10033_6832col_10033_48157
Wodke, Judith A H
Puchałka, Jacek
Lluch-Senar, Maria
Marcos, Josep
Yus, Eva
Godinho, Miguel
Gutiérrez-Gallego, Ricardo
dos Santos, Vitor A P Martins
Serrano, Luis
Klipp, Edda
Maier, Tobias
EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Barcelona, Spain.
2013-10-08T13:50:36Z
2013-10-08T13:50:36Z
2013
Dissecting the energy metabolism in Mycoplasma pneumoniae through genome-scale metabolic modeling. 2013, 9:653 Mol. Syst. Biol.
1744-4292
23549481
10.1038/msb.2013.6
http://hdl.handle.net/10033/302985
Molecular systems biology
Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint-based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time-dependent changes, albeit using a static model. By performing an in silico knock-out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.
en
Archived with thanks to Molecular systems biology
Computer Simulation
Energy Metabolism
Gene Expression Regulation, Bacterial
Genome, Bacterial
Metabolic Networks and Pathways
Models, Biological
Mutation
Mycoplasma pneumoniae
Dissecting the energy metabolism in Mycoplasma pneumoniae through genome-scale metabolic modeling.
Article2018-06-13T07:35:14ZMycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint-based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time-dependent changes, albeit using a static model. By performing an in silico knock-out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.oai:repository.helmholtz-hzi.de:10033/3048122019-08-30T11:33:30Zcom_10033_6832col_10033_6833
Hofmann, Julia
Heider, Christine
Li, Wei
Krausze, Joern
Roessle, Manfred
Wilharm, Gottfried
Robert Koch-Institute, Wernigerode Branch, Burgstr. 37, D-38855 Wernigerode, Germany.
2013-10-31T12:11:40Z
2013-10-31T12:11:40Z
2013-04
Recombinant production of Yersinia enterocolitica pyruvate kinase isoenzymes PykA and PykF. 2013, 88 (2):243-7 Protein Expr. Purif.
1096-0279
23384479
10.1016/j.pep.2013.01.010
http://hdl.handle.net/10033/304812
Protein expression and purification
The glycolytic enzyme pyruvate kinase (PK) generates ATP from ADP through substrate-level phosphorylation powered by the conversion of phosphoenolpyruvate to pyruvate. In contrast to other bacteria, Enterobacteriaceae, such as pathogenic yersiniae, harbour two pyruvate kinases encoded by pykA and pykF. The individual roles of these isoenzymes are poorly understood. In an attempt to make the Yersinia enterocolitica pyruvate kinases PykA and PykF amenable to structural and functional characterisation, we produced them untagged in Escherichia coli and purified them to near homogeneity through a combination of ion exchange and size exclusion chromatography, yielding more than 180 mg per litre of batch culture. The solution structure of PykA and PykF was analysed through small angle X-ray scattering which revealed the formation of PykA and PykF tetramers and confirmed the binding of the allosteric effector fructose-1,6-bisphosphate (FBP) to PykF but not to PykA.
en
Archived with thanks to Protein expression and purification
Chromatography, Gel
Chromatography, Ion Exchange
Escherichia coli
Gene Expression
Genetic Vectors
Isoenzymes
Models, Molecular
Protein Multimerization
Pyruvate Kinase
Recombinant Proteins
Scattering, Small Angle
X-Ray Diffraction
Yersinia enterocolitica
Recombinant production of Yersinia enterocolitica pyruvate kinase isoenzymes PykA and PykF.
Article2018-06-12T21:24:57ZThe glycolytic enzyme pyruvate kinase (PK) generates ATP from ADP through substrate-level phosphorylation powered by the conversion of phosphoenolpyruvate to pyruvate. In contrast to other bacteria, Enterobacteriaceae, such as pathogenic yersiniae, harbour two pyruvate kinases encoded by pykA and pykF. The individual roles of these isoenzymes are poorly understood. In an attempt to make the Yersinia enterocolitica pyruvate kinases PykA and PykF amenable to structural and functional characterisation, we produced them untagged in Escherichia coli and purified them to near homogeneity through a combination of ion exchange and size exclusion chromatography, yielding more than 180 mg per litre of batch culture. The solution structure of PykA and PykF was analysed through small angle X-ray scattering which revealed the formation of PykA and PykF tetramers and confirmed the binding of the allosteric effector fructose-1,6-bisphosphate (FBP) to PykF but not to PykA.oai:repository.helmholtz-hzi.de:10033/3061672019-08-30T11:37:44Zcom_10033_305061com_10033_6832col_10033_305062
Hellert, Jan
Weidner-Glunde, Magdalena
Krausze, Joern
Richter, Ulrike
Adler, Heiko
Fedorov, Roman
Pietrek, Marcel
Rückert, Jessica
Ritter, Christiane
Schulz, Thomas F
Lührs, Thorsten
Department of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
2013-12-03T13:03:08Z
2013-12-03T13:03:08Z
2013-10
A Structural Basis for BRD2/4-Mediated Host Chromatin Interaction and Oligomer Assembly of Kaposi Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus LANA Proteins. 2013, 9 (10):e1003640 PLoS Pathog.
1553-7374
24146614
10.1371/journal.ppat.1003640
http://hdl.handle.net/10033/306167
PLoS pathogens
Kaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related γ2-herpesvirus frequently used as a model to study the biology of γ-herpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed 'LANA speckles', which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of γ2-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA 'nuclear speckles' and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence.
en
Archived with thanks to PLoS pathogens
A Structural Basis for BRD2/4-Mediated Host Chromatin Interaction and Oligomer Assembly of Kaposi Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus LANA Proteins.
Article2018-06-12T16:42:14ZKaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related γ2-herpesvirus frequently used as a model to study the biology of γ-herpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed 'LANA speckles', which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of γ2-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA 'nuclear speckles' and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence.oai:repository.helmholtz-hzi.de:10033/3160362019-08-30T11:26:42Zcom_10033_271853com_10033_6832col_10033_271872
Vahokoski, Juha
Bhargav, Saligram Prabhakar
Desfosses, Ambroise
Andreadaki, Maria
Kumpula, Esa-Pekka
Martinez, Silvia Muñico
Ignatev, Alexander
Lepper, Simone
Frischknecht, Friedrich
Sidén-Kiamos, Inga
Sachse, Carsten
Kursula, Inari
Strucural biology of the cytoskeleton, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany.
2014-04-22T12:36:37Z
2014-04-22T12:36:37Z
2014-04
Structural differences explain diverse functions of Plasmodium actins. 2014, 10 (4):e1004091 PLoS Pathog.
1553-7374
24743229
10.1371/journal.ppat.1004091
http://hdl.handle.net/10033/316036
PLoS pathogens
Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties.
en
Archived with thanks to PLoS pathogens
Structural differences explain diverse functions of Plasmodium actins.
Article2018-06-12T23:16:51ZActins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties.oai:repository.helmholtz-hzi.de:10033/3164572019-08-30T11:26:42Zcom_10033_271853com_10033_6832col_10033_271872
Han, Huijong
Kursula, Petri
2014-05-02T14:10:50Z
2014-05-02T14:10:50Z
2013-07
Preliminary crystallographic analysis of the N-terminal PDZ-like domain of periaxin, an abundant peripheral nerve protein linked to human neuropathies. 2013, 69 (Pt 7):804-8 Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
1744-3091
23832213
10.1107/S1744309113016266
http://hdl.handle.net/10033/316457
Acta crystallographica. Section F, Structural biology and crystallization communications
Periaxin (PRX) is an abundant protein in peripheral nerves and contains a predicted PDZ-like domain at its N-terminus. The large isoform, L-PRX, is required for the maintenance of myelin in the peripheral nervous system and its defects cause neurological disease. Here, the human periaxin PDZ-like domain was crystallized and X-ray diffraction data were collected to 2.85 Å resolution using synchrotron radiation. The crystal belonged to the primitive hexagonal space group P3121 or P3221, with unit-cell parameters a = b = 80.6, c = 81.0 Å, γ = 120° and either two or three molecules in the asymmetric unit. The structure of PRX will shed light on its poorly characterized function in the nervous system.
en
Archived with thanks to Acta crystallographica. Section F, Structural biology and crystallization communications
Amino Acid Sequence
Crystallization
Crystallography, X-Ray
Humans
Membrane Proteins
Molecular Sequence Data
PDZ Domains
Peripheral Nerves
Peripheral Nervous System Diseases
Recombinant Proteins
Sequence Homology, Amino Acid
Synchrotrons
Preliminary crystallographic analysis of the N-terminal PDZ-like domain of periaxin, an abundant peripheral nerve protein linked to human neuropathies.
Article2018-06-13T07:23:18ZPeriaxin (PRX) is an abundant protein in peripheral nerves and contains a predicted PDZ-like domain at its N-terminus. The large isoform, L-PRX, is required for the maintenance of myelin in the peripheral nervous system and its defects cause neurological disease. Here, the human periaxin PDZ-like domain was crystallized and X-ray diffraction data were collected to 2.85 Å resolution using synchrotron radiation. The crystal belonged to the primitive hexagonal space group P3121 or P3221, with unit-cell parameters a = b = 80.6, c = 81.0 Å, γ = 120° and either two or three molecules in the asymmetric unit. The structure of PRX will shed light on its poorly characterized function in the nervous system.oai:repository.helmholtz-hzi.de:10033/3173862019-08-30T11:30:58Zcom_10033_48156com_10033_6832col_10033_48157
Barbier, Mariette
Damron, F Heath
Bielecki, Piotr
Suárez-Diez, María
Puchałka, Jacek
Albertí, Sebastian
Dos Santos, Vitor Martins
Goldberg, Joanna B
Synthetic and Systems Biology Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany
2014-05-23T12:40:54Z
2014-05-23T12:40:54Z
2014
From the environment to the host: re-wiring of the transcriptome of Pseudomonas aeruginosa from 22°C to 37°C. 2014, 9 (2):e89941 PLoS ONE
1932-6203
24587139
10.1371/journal.pone.0089941
http://hdl.handle.net/10033/317386
PloS one
Pseudomonas aeruginosa is a highly versatile opportunistic pathogen capable of colonizing multiple ecological niches. This bacterium is responsible for a wide range of both acute and chronic infections in a variety of hosts. The success of this microorganism relies on its ability to adapt to environmental changes and re-program its regulatory and metabolic networks. The study of P. aeruginosa adaptation to temperature is crucial to understanding the pathogenesis upon infection of its mammalian host. We examined the effects of growth temperature on the transcriptome of the P. aeruginosa PAO1. Microarray analysis of PAO1 grown in Lysogeny broth at mid-exponential phase at 22°C and 37°C revealed that temperature changes are responsible for the differential transcriptional regulation of 6.4% of the genome. Major alterations were observed in bacterial metabolism, replication, and nutrient acquisition. Quorum-sensing and exoproteins secreted by type I, II, and III secretion systems, involved in the adaptation of P. aeruginosa to the mammalian host during infection, were up-regulated at 37°C compared to 22°C. Genes encoding arginine degradation enzymes were highly up-regulated at 22°C, together with the genes involved in the synthesis of pyoverdine. However, genes involved in pyochelin biosynthesis were up-regulated at 37°C. We observed that the changes in expression of P. aeruginosa siderophores correlated to an overall increase in Fe²⁺ extracellular concentration at 37°C and a peak in Fe³⁺ extracellular concentration at 22°C. This suggests a distinct change in iron acquisition strategies when the bacterium switches from the external environment to the host. Our work identifies global changes in bacterial metabolism and nutrient acquisition induced by growth at different temperatures. Overall, this study identifies factors that are regulated in genome-wide adaptation processes and discusses how this life-threatening pathogen responds to temperature.
en
Archived with thanks to PloS one
From the environment to the host: re-wiring of the transcriptome of Pseudomonas aeruginosa from 22°C to 37°C.
Article2018-06-13T16:58:28ZPseudomonas aeruginosa is a highly versatile opportunistic pathogen capable of colonizing multiple ecological niches. This bacterium is responsible for a wide range of both acute and chronic infections in a variety of hosts. The success of this microorganism relies on its ability to adapt to environmental changes and re-program its regulatory and metabolic networks. The study of P. aeruginosa adaptation to temperature is crucial to understanding the pathogenesis upon infection of its mammalian host. We examined the effects of growth temperature on the transcriptome of the P. aeruginosa PAO1. Microarray analysis of PAO1 grown in Lysogeny broth at mid-exponential phase at 22°C and 37°C revealed that temperature changes are responsible for the differential transcriptional regulation of 6.4% of the genome. Major alterations were observed in bacterial metabolism, replication, and nutrient acquisition. Quorum-sensing and exoproteins secreted by type I, II, and III secretion systems, involved in the adaptation of P. aeruginosa to the mammalian host during infection, were up-regulated at 37°C compared to 22°C. Genes encoding arginine degradation enzymes were highly up-regulated at 22°C, together with the genes involved in the synthesis of pyoverdine. However, genes involved in pyochelin biosynthesis were up-regulated at 37°C. We observed that the changes in expression of P. aeruginosa siderophores correlated to an overall increase in Fe²⁺ extracellular concentration at 37°C and a peak in Fe³⁺ extracellular concentration at 22°C. This suggests a distinct change in iron acquisition strategies when the bacterium switches from the external environment to the host. Our work identifies global changes in bacterial metabolism and nutrient acquisition induced by growth at different temperatures. Overall, this study identifies factors that are regulated in genome-wide adaptation processes and discusses how this life-threatening pathogen responds to temperature.oai:repository.helmholtz-hzi.de:10033/3207952019-08-30T11:31:23Zcom_10033_271853com_10033_6832col_10033_271872
Lehtimäki, Mari
Laulumaa, Saara
Ruskamo, Salla
Kursula, Petri
2014-06-11T13:22:33Z
2014-06-11T13:22:33Z
2012-11-01
Production and crystallization of a panel of structure-based mutants of the human myelin peripheral membrane protein P2. 2012, 68 (Pt 11):1359-62 Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
1744-3091
23143249
10.1107/S1744309112039036
http://hdl.handle.net/10033/320795
Acta crystallographica. Section F, Structural biology and crystallization communications
The myelin sheath is a multilayered membrane that surrounds and insulates axons in the nervous system. One of the proteins specific to the peripheral nerve myelin is P2, a protein that is able to stack lipid bilayers. With the goal of obtaining detailed information on the structure-function relationship of P2, 14 structure-based mutated variants of human P2 were generated and produced. The mutants were designed to potentially affect the binding of lipid bilayers by P2. All mutated variants were also crystallized and preliminary crystallographic data are presented. The structural data from the mutants will be combined with diverse functional assays in order to elucidate the fine details of P2 function at the molecular level.
en
Archived with thanks to Acta crystallographica. Section F, Structural biology and crystallization communications
Amino Acid Substitution
Crystallization
Escherichia coli
Humans
Hydrophobic and Hydrophilic Interactions
Models, Molecular
Mutagenesis, Site-Directed
Myelin P2 Protein
Protein Structure, Secondary
Protein Structure, Tertiary
X-Ray Diffraction
Production and crystallization of a panel of structure-based mutants of the human myelin peripheral membrane protein P2.
Article2018-06-13T19:36:19ZThe myelin sheath is a multilayered membrane that surrounds and insulates axons in the nervous system. One of the proteins specific to the peripheral nerve myelin is P2, a protein that is able to stack lipid bilayers. With the goal of obtaining detailed information on the structure-function relationship of P2, 14 structure-based mutated variants of human P2 were generated and produced. The mutants were designed to potentially affect the binding of lipid bilayers by P2. All mutated variants were also crystallized and preliminary crystallographic data are presented. The structural data from the mutants will be combined with diverse functional assays in order to elucidate the fine details of P2 function at the molecular level.oai:repository.helmholtz-hzi.de:10033/3234342019-08-30T11:35:39Zcom_10033_271853com_10033_6832col_10033_271872
Ruskamo, Salla
Chukhlieb, Maryna
Vahokoski, Juha
Bhargav, Saligram Prabhakar
Liang, Fengyi
Kursula, Inari
Kursula, Petri
2014-07-18T14:30:54Z
2014-07-18T14:30:54Z
2012
Juxtanodin is an intrinsically disordered F-actin-binding protein. 2012, 2:899 Sci Rep
2045-2322
23198089
10.1038/srep00899
http://hdl.handle.net/10033/323434
Scientific reports
Juxtanodin, also called ermin, is an F-actin-binding protein expressed by oligodendrocytes, the myelin-forming cells of the central nervous system. While juxtanodin carries a short conserved F-actin-binding segment at its C terminus, it otherwise shares no similarity with known protein sequences. We carried out a structural characterization of recombinant juxtanodin in solution. Juxtanodin turned out to be intrinsically disordered, as evidenced by conventional and synchrotron radiation CD spectroscopy. Small-angle X-ray scattering indicated that juxtanodin is a monomeric, highly elongated, unfolded molecule. Ensemble optimization analysis of the data suggested also the presence of more compact forms of juxtanodin. The C terminus was a strict requirement for co-sedimentation of juxtanodin with microfilaments, but juxtanodin had only mild effects on actin polymerization. The disordered nature of juxtanodin may predict functions as a protein interaction hub, although F-actin is its only currently known binding partner.
en
Archived with thanks to Scientific reports
Actins
Algorithms
Amino Acid Sequence
Animals
Carrier Proteins
Circular Dichroism
Microfilament Proteins
Microscopy, Electron
Molecular Sequence Data
Muscle, Skeletal
Protein Binding
Protein Structure, Secondary
Rats
Recombinant Proteins
Scattering, Small Angle
Sequence Homology, Amino Acid
Software
Solutions
Swine
X-Ray Diffraction
Juxtanodin is an intrinsically disordered F-actin-binding protein.
Article2018-06-12T17:59:28ZJuxtanodin, also called ermin, is an F-actin-binding protein expressed by oligodendrocytes, the myelin-forming cells of the central nervous system. While juxtanodin carries a short conserved F-actin-binding segment at its C terminus, it otherwise shares no similarity with known protein sequences. We carried out a structural characterization of recombinant juxtanodin in solution. Juxtanodin turned out to be intrinsically disordered, as evidenced by conventional and synchrotron radiation CD spectroscopy. Small-angle X-ray scattering indicated that juxtanodin is a monomeric, highly elongated, unfolded molecule. Ensemble optimization analysis of the data suggested also the presence of more compact forms of juxtanodin. The C terminus was a strict requirement for co-sedimentation of juxtanodin with microfilaments, but juxtanodin had only mild effects on actin polymerization. The disordered nature of juxtanodin may predict functions as a protein interaction hub, although F-actin is its only currently known binding partner.oai:repository.helmholtz-hzi.de:10033/3243472019-08-30T11:36:04Zcom_10033_48156com_10033_6832col_10033_48157
Fang, Kechi
Zhao, Hansheng
Sun, Changyue
Lam, Carolyn M C
Chang, Suhua
Zhang, Kunlin
Panda, Gurudutta
Godinho, Miguel
Martins dos Santos, Vítor A P
Wang, Jing
2014-08-07T13:05:02Z
2014-08-07T13:05:02Z
2011
Exploring the metabolic network of the epidemic pathogen Burkholderia cenocepacia J2315 via genome-scale reconstruction. 2011, 5:83 BMC Syst Biol
1752-0509
21609491
10.1186/1752-0509-5-83
http://hdl.handle.net/10033/324347
BMC systems biology
Burkholderia cenocepacia is a threatening nosocomial epidemic pathogen in patients with cystic fibrosis (CF) or a compromised immune system. Its high level of antibiotic resistance is an increasing concern in treatments against its infection. Strain B. cenocepacia J2315 is the most infectious isolate from CF patients. There is a strong demand to reconstruct a genome-scale metabolic network of B. cenocepacia J2315 to systematically analyze its metabolic capabilities and its virulence traits, and to search for potential clinical therapy targets.
en
Archived with thanks to BMC systems biology
Anti-Bacterial Agents
Biomass
Burkholderia Infections
Burkholderia cenocepacia
Catalysis
Computational Biology
Drug Resistance, Bacterial
Fatty Acids
Genome
Genome, Bacterial
Humans
Lipopolysaccharides
Models, Biological
Models, Statistical
Phenotype
Systems Biology
Exploring the metabolic network of the epidemic pathogen Burkholderia cenocepacia J2315 via genome-scale reconstruction.
Article2018-06-13T14:09:05ZBurkholderia cenocepacia is a threatening nosocomial epidemic pathogen in patients with cystic fibrosis (CF) or a compromised immune system. Its high level of antibiotic resistance is an increasing concern in treatments against its infection. Strain B. cenocepacia J2315 is the most infectious isolate from CF patients. There is a strong demand to reconstruct a genome-scale metabolic network of B. cenocepacia J2315 to systematically analyze its metabolic capabilities and its virulence traits, and to search for potential clinical therapy targets.oai:repository.helmholtz-hzi.de:10033/3244832019-08-30T11:36:05Zcom_10033_271853com_10033_6832col_10033_271872
Patel, Ashok K
Singh, Vijay K
Bergmann, Ulrich
Jagannadham, Medicherla V
Kursula, Petri
2014-08-08T10:42:32Z
2014-08-08T10:42:32Z
2011-05-01
Purification, crystallization and preliminary X-ray crystallographic analysis of MIL, a glycosylated jacalin-related lectin from mulberry (Morus indica) latex. 2011, 67 (Pt 5):608-12 Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
1744-3091
21543873
10.1107/S174430911101013X
http://hdl.handle.net/10033/324483
Acta crystallographica. Section F, Structural biology and crystallization communications
A quantitatively major protein has been purified from the latex of Morus indica. The purified previously uncharacterized protein, M. indica lectin (MIL), was further shown to be a glycosylated tetramer and belongs to the family of jacalin-related lectins. Crystallization of MIL was also accomplished and the tetragonal crystals diffracted synchrotron X-rays to a resolution of 2.8 Å.
en
Archived with thanks to Acta crystallographica. Section F, Structural biology and crystallization communications
Amino Acid Sequence
Crystallization
Crystallography, X-Ray
Glycosylation
Molecular Sequence Data
Morus
Plant Lectins
Sequence Alignment
Purification, crystallization and preliminary X-ray crystallographic analysis of MIL, a glycosylated jacalin-related lectin from mulberry (Morus indica) latex.
Article2018-06-13T00:25:20ZA quantitatively major protein has been purified from the latex of Morus indica. The purified previously uncharacterized protein, M. indica lectin (MIL), was further shown to be a glycosylated tetramer and belongs to the family of jacalin-related lectins. Crystallization of MIL was also accomplished and the tetragonal crystals diffracted synchrotron X-rays to a resolution of 2.8 Å.oai:repository.helmholtz-hzi.de:10033/3328422019-08-30T11:27:46Zcom_10033_271853com_10033_6832col_10033_271872
Myllykoski, Matti
Kursula, Petri
2014-10-16T13:46:51Z
2014-10-16T13:46:51Z
2010
Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath. 2010, 3:12 BMC Res Notes
1756-0500
20180985
10.1186/1756-0500-3-12
http://hdl.handle.net/10033/332842
BMC research notes
2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enigmatic enzyme specifically expressed at high levels in the vertebrate myelin sheath, whose function and physiological substrates are unknown. The protein consists of two domains: an uncharacterized N-terminal domain with little homology to other proteins, and a C-terminal phosphodiesterase domain.
en
Archived with thanks to BMC research notes
Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath.
Article2018-06-12T23:36:02Z2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enigmatic enzyme specifically expressed at high levels in the vertebrate myelin sheath, whose function and physiological substrates are unknown. The protein consists of two domains: an uncharacterized N-terminal domain with little homology to other proteins, and a C-terminal phosphodiesterase domain.oai:repository.helmholtz-hzi.de:10033/3390362019-08-30T11:36:32Zcom_10033_48156com_10033_6832col_10033_48157
Porcar, Manuel
Danchin, Antoine
de Lorenzo, Victor
Dos Santos, Vitor A
Krasnogor, Natalio
Rasmussen, Steen
Moya, Andrés
2015-01-29T12:40:39Z
2015-01-29T12:40:39Z
2011-06
The ten grand challenges of synthetic life. 2011, 5 (1-2):1-9 Syst Synth Biol
1872-5333
21949672
10.1007/s11693-011-9084-5
http://hdl.handle.net/10033/339036
Systems and synthetic biology
The construction of artificial life is one of the main scientific challenges of the Synthetic Biology era. Advances in DNA synthesis and a better understanding of regulatory processes make the goal of constructing the first artificial cell a realistic possibility. This would be both a fundamental scientific milestone and a starting point of a vast range of applications, from biofuel production to drug design. However, several major issues might hamper the objective of achieving an artificial cell. From the bottom-up to the selection-based strategies, this work encompasses the ten grand challenges synthetic biologists will have to be aware of in order to cope with the task of creating life in the lab.
en
The ten grand challenges of synthetic life.
Article2018-06-13T20:02:26ZThe construction of artificial life is one of the main scientific challenges of the Synthetic Biology era. Advances in DNA synthesis and a better understanding of regulatory processes make the goal of constructing the first artificial cell a realistic possibility. This would be both a fundamental scientific milestone and a starting point of a vast range of applications, from biofuel production to drug design. However, several major issues might hamper the objective of achieving an artificial cell. From the bottom-up to the selection-based strategies, this work encompasses the ten grand challenges synthetic biologists will have to be aware of in order to cope with the task of creating life in the lab.oai:repository.helmholtz-hzi.de:10033/3443852019-08-30T11:37:23Zcom_10033_271853com_10033_6832col_10033_271872
Singh, Bishal K
Sattler, Julia M
Chatterjee, Moon
Huttu, Jani
Schüler, Herwig
Kursula, Inari
2015-02-12T13:11:48Z
2015-02-12T13:11:48Z
2011-08-12
Crystal structures explain functional differences in the two actin depolymerization factors of the malaria parasite. 2011, 286 (32):28256-64 J. Biol. Chem.
1083-351X
21832095
10.1074/jbc.M111.211730
http://hdl.handle.net/10033/344385
The Journal of biological chemistry
Apicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multifaceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here, we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.
en
Crystallography, X-Ray
Destrin
Plasmodium berghei
Plasmodium falciparum
Protein Structure, Tertiary
Protozoan Proteins
Species Specificity
Structure-Activity Relationship
Crystal structures explain functional differences in the two actin depolymerization factors of the malaria parasite.
Article2018-06-13T19:29:00ZApicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multifaceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here, we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.oai:repository.helmholtz-hzi.de:10033/3444102019-08-30T11:25:43Zcom_10033_271853com_10033_6832col_10033_271872
Patel, Ashok K
Yadav, Ravi P
Majava, Viivi
Kursula, Inari
Kursula, Petri
2015-02-12T12:38:59Z
2015-02-12T12:38:59Z
2011-06-10
Structure of the dimeric autoinhibited conformation of DAPK2, a pro-apoptotic protein kinase. 2011, 409 (3):369-83 J. Mol. Biol.
1089-8638
21497605
10.1016/j.jmb.2011.03.065
http://hdl.handle.net/10033/344410
Journal of molecular biology
The death-associated protein kinase (DAPK) family has been characterized as a group of pro-apoptotic serine/threonine kinases that share specific structural features in their catalytic kinase domain. Two of the DAPK family members, DAPK1 and DAPK2, are calmodulin-dependent protein kinases that are regulated by oligomerization, calmodulin binding, and autophosphorylation. In this study, we have determined the crystal and solution structures of murine DAPK2 in the presence of the autoinhibitory domain, with and without bound nucleotides in the active site. The crystal structure shows dimers of DAPK2 in a conformation that is not permissible for protein substrate binding. Two different conformations were seen in the active site upon the introduction of nucleotide ligands. The monomeric and dimeric forms of DAPK2 were further analyzed for solution structure, and the results indicate that the dimers of DAPK2 are indeed formed through the association of two apposed catalytic domains, as seen in the crystal structure. The structures can be further used to build a model for DAPK2 autophosphorylation and to compare with closely related kinases, of which especially DAPK1 is an actively studied drug target. Our structures also provide a model for both homodimerization and heterodimerization of the catalytic domain between members of the DAPK family. The fingerprint of the DAPK family, the basic loop, plays a central role in the dimerization of the kinase domain.
en
Animals
Apoptosis
Apoptosis Regulatory Proteins
Calcium-Calmodulin-Dependent Protein Kinases
Catalytic Domain
Crystallography, X-Ray
Death-Associated Protein Kinases
Dimerization
Mice
Models, Molecular
Phosphorylation
Protein Conformation
Protein Interaction Domains and Motifs
Sequence Homology, Amino Acid
Structure of the dimeric autoinhibited conformation of DAPK2, a pro-apoptotic protein kinase.
Article2018-06-13T01:01:45ZThe death-associated protein kinase (DAPK) family has been characterized as a group of pro-apoptotic serine/threonine kinases that share specific structural features in their catalytic kinase domain. Two of the DAPK family members, DAPK1 and DAPK2, are calmodulin-dependent protein kinases that are regulated by oligomerization, calmodulin binding, and autophosphorylation. In this study, we have determined the crystal and solution structures of murine DAPK2 in the presence of the autoinhibitory domain, with and without bound nucleotides in the active site. The crystal structure shows dimers of DAPK2 in a conformation that is not permissible for protein substrate binding. Two different conformations were seen in the active site upon the introduction of nucleotide ligands. The monomeric and dimeric forms of DAPK2 were further analyzed for solution structure, and the results indicate that the dimers of DAPK2 are indeed formed through the association of two apposed catalytic domains, as seen in the crystal structure. The structures can be further used to build a model for DAPK2 autophosphorylation and to compare with closely related kinases, of which especially DAPK1 is an actively studied drug target. Our structures also provide a model for both homodimerization and heterodimerization of the catalytic domain between members of the DAPK family. The fingerprint of the DAPK family, the basic loop, plays a central role in the dimerization of the kinase domain.oai:repository.helmholtz-hzi.de:10033/5612262019-08-30T11:28:23Zcom_10033_6835com_10033_6832col_10033_6836
Stoppok, W
Rapp, P
Wagner, F
Gesellschaft fur Biotechnologische Forschung mbH, D-38124 Braunschweig, Germany.
2015-07-30T09:13:38Z
2015-07-30T09:13:38Z
1982-07
Formation, Location, and Regulation of Endo-1,4-beta-Glucanases and beta-Glucosidases from Cellulomonas uda. 1982, 44 (1):44-53 Appl. Environ. Microbiol.
0099-2240
16346067
http://hdl.handle.net/10033/561226
Applied and environmental microbiology
The formation and location of endo-1,4-beta-glucanases and beta-glucosidases were studied in cultures of Cellulomonas uda grown on microcrystalline cellulose, carboxymethyl cellulose, printed newspaper, and some mono- or disaccharides. Endo-1,4-Glucanases were found to be extracellular, but a very small amount of cell-bound endo-1,4-beta-glucanase was considered to be the basal endoglucanase level of the cells. The formation of extracellular endo-1,4-beta-glucanases was induced by cellobiose and repressed by glucose. Extracellular endoglucanase activity was inhibited by cellobiose but not by glucose. beta-Glucosidases, on the other hand, were formed constitutively and found to be cell bound. beta-Glucosidase activity was inhibited noncompetitively by glucose. Some characteristics such as the optimal pH for and the thermostability of the endoglucanases and beta-glucosidases and the end products of cellulose degradation were determined.
en
Formation, Location, and Regulation of Endo-1,4-beta-Glucanases and beta-Glucosidases from Cellulomonas uda.
Article2018-06-13T15:12:46ZThe formation and location of endo-1,4-beta-glucanases and beta-glucosidases were studied in cultures of Cellulomonas uda grown on microcrystalline cellulose, carboxymethyl cellulose, printed newspaper, and some mono- or disaccharides. Endo-1,4-Glucanases were found to be extracellular, but a very small amount of cell-bound endo-1,4-beta-glucanase was considered to be the basal endoglucanase level of the cells. The formation of extracellular endo-1,4-beta-glucanases was induced by cellobiose and repressed by glucose. Extracellular endoglucanase activity was inhibited by cellobiose but not by glucose. beta-Glucosidases, on the other hand, were formed constitutively and found to be cell bound. beta-Glucosidase activity was inhibited noncompetitively by glucose. Some characteristics such as the optimal pH for and the thermostability of the endoglucanases and beta-glucosidases and the end products of cellulose degradation were determined.oai:repository.helmholtz-hzi.de:10033/6210442018-12-20T01:42:22Zcom_10033_6832col_10033_6833
Sun, Jibin
Lu, Xin
Rinas, Ursula
Ping Zeng, An
2017-08-04T08:46:09Z
2017-08-04T08:46:09Z
2007-09-04
2015-09-04T08:23:05Z
Genome Biology. 2007 Sep 04;8(9):R182
http://dx.doi.org/10.1186/gb-2007-8-9-r182
http://hdl.handle.net/10033/621044
Abstract Background Aspergillus niger is an important industrial microorganism for the production of both metabolites, such as citric acid, and proteins, such as fungal enzymes or heterologous proteins. Despite its extensive industrial applications, the genetic inventory of this fungus is only partially understood. The recently released genome sequence opens a new horizon for both scientific studies and biotechnological applications. Results Here, we present the first genome-scale metabolic network for A. niger and an in-depth genomic comparison of this species to seven other fungi to disclose its metabolic peculiarities. The raw genomic sequences of A. niger ATCC 9029 were first annotated. The reconstructed metabolic network is based on the annotation of two A. niger genomes, CBS 513.88 and ATCC 9029, including enzymes with 988 unique EC numbers, 2,443 reactions and 2,349 metabolites. More than 1,100 enzyme-coding genes are unique to A. niger in comparison to the other seven fungi. For example, we identified additional copies of genes such as those encoding alternative mitochondrial oxidoreductase and citrate synthase in A. niger, which might contribute to the high citric acid production efficiency of this species. Moreover, nine genes were identified as encoding enzymes with EC numbers exclusively found in A. niger, mostly involved in the biosynthesis of complex secondary metabolites and degradation of aromatic compounds. Conclusion The genome-level reconstruction of the metabolic network and genome-based metabolic comparison disclose peculiarities of A. niger highly relevant to its biotechnological applications and should contribute to future rational metabolic design and systems biology studies of this black mold and related species.
Metabolic peculiarities of Aspergillus niger disclosed by comparative metabolic genomics
Journal Article
en
Sun et al..2018-06-12T16:52:19ZAbstract
Background
Aspergillus niger is an important industrial microorganism for the production of both metabolites, such as citric acid, and proteins, such as fungal enzymes or heterologous proteins. Despite its extensive industrial applications, the genetic inventory of this fungus is only partially understood. The recently released genome sequence opens a new horizon for both scientific studies and biotechnological applications.
Results
Here, we present the first genome-scale metabolic network for A. niger and an in-depth genomic comparison of this species to seven other fungi to disclose its metabolic peculiarities. The raw genomic sequences of A. niger ATCC 9029 were first annotated. The reconstructed metabolic network is based on the annotation of two A. niger genomes, CBS 513.88 and ATCC 9029, including enzymes with 988 unique EC numbers, 2,443 reactions and 2,349 metabolites. More than 1,100 enzyme-coding genes are unique to A. niger in comparison to the other seven fungi. For example, we identified additional copies of genes such as those encoding alternative mitochondrial oxidoreductase and citrate synthase in A. niger, which might contribute to the high citric acid production efficiency of this species. Moreover, nine genes were identified as encoding enzymes with EC numbers exclusively found in A. niger, mostly involved in the biosynthesis of complex secondary metabolites and degradation of aromatic compounds.
Conclusion
The genome-level reconstruction of the metabolic network and genome-based metabolic comparison disclose peculiarities of A. niger highly relevant to its biotechnological applications and should contribute to future rational metabolic design and systems biology studies of this black mold and related species.oai:repository.helmholtz-hzi.de:10033/6206832019-08-30T11:35:39Zcom_10033_48156com_10033_6832col_10033_48157
Fang, Kechi
Zhao, Hansheng
Sun, Changyue
Lam, Carolyn M C
Chang, Suhua
Zhang, Kunlin
Panda, Gurudutta
Godinho, Miguel
Martins dos Santos, Vítor A P
Wang, Jing
2017-01-06T10:24:53Z
2017-01-06T10:24:53Z
2011-05-25
2015-09-04T08:24:26Z
BMC Systems Biology. 2011 May 25;5(1):83
http://dx.doi.org/10.1186/1752-0509-5-83
http://hdl.handle.net/10033/620683
Abstract Background Burkholderia cenocepacia is a threatening nosocomial epidemic pathogen in patients with cystic fibrosis (CF) or a compromised immune system. Its high level of antibiotic resistance is an increasing concern in treatments against its infection. Strain B. cenocepacia J2315 is the most infectious isolate from CF patients. There is a strong demand to reconstruct a genome-scale metabolic network of B. cenocepacia J2315 to systematically analyze its metabolic capabilities and its virulence traits, and to search for potential clinical therapy targets. Results We reconstructed the genome-scale metabolic network of B. cenocepacia J2315. An iterative reconstruction process led to the establishment of a robust model, iKF1028, which accounts for 1,028 genes, 859 internal reactions, and 834 metabolites. The model iKF1028 captures important metabolic capabilities of B. cenocepacia J2315 with a particular focus on the biosyntheses of key metabolic virulence factors to assist in understanding the mechanism of disease infection and identifying potential drug targets. The model was tested through BIOLOG assays. Based on the model, the genome annotation of B. cenocepacia J2315 was refined and 24 genes were properly re-annotated. Gene and enzyme essentiality were analyzed to provide further insights into the genome function and architecture. A total of 45 essential enzymes were identified as potential therapeutic targets. Conclusions As the first genome-scale metabolic network of B. cenocepacia J2315, iKF1028 allows a systematic study of the metabolic properties of B. cenocepacia and its key metabolic virulence factors affecting the CF community. The model can be used as a discovery tool to design novel drugs against diseases caused by this notorious pathogen.
Exploring the metabolic network of the epidemic pathogen Burkholderia cenocepacia J2315 via genome-scale reconstruction
Journal Article
en
Fang et al; licensee BioMed Central Ltd.2018-06-12T23:46:44ZAbstract
Background
Burkholderia cenocepacia is a threatening nosocomial epidemic pathogen in patients with cystic fibrosis (CF) or a compromised immune system. Its high level of antibiotic resistance is an increasing concern in treatments against its infection. Strain B. cenocepacia J2315 is the most infectious isolate from CF patients. There is a strong demand to reconstruct a genome-scale metabolic network of B. cenocepacia J2315 to systematically analyze its metabolic capabilities and its virulence traits, and to search for potential clinical therapy targets.
Results
We reconstructed the genome-scale metabolic network of B. cenocepacia J2315. An iterative reconstruction process led to the establishment of a robust model, iKF1028, which accounts for 1,028 genes, 859 internal reactions, and 834 metabolites. The model iKF1028 captures important metabolic capabilities of B. cenocepacia J2315 with a particular focus on the biosyntheses of key metabolic virulence factors to assist in understanding the mechanism of disease infection and identifying potential drug targets. The model was tested through BIOLOG assays. Based on the model, the genome annotation of B. cenocepacia J2315 was refined and 24 genes were properly re-annotated. Gene and enzyme essentiality were analyzed to provide further insights into the genome function and architecture. A total of 45 essential enzymes were identified as potential therapeutic targets.
Conclusions
As the first genome-scale metabolic network of B. cenocepacia J2315, iKF1028 allows a systematic study of the metabolic properties of B. cenocepacia and its key metabolic virulence factors affecting the CF community. The model can be used as a discovery tool to design novel drugs against diseases caused by this notorious pathogen.oai:repository.helmholtz-hzi.de:10033/6206962019-08-30T11:26:13Zcom_10033_6835com_10033_6832col_10033_6836
Sharma, Alok
Bruns, Karsten
Röder, René
Henklein, Peter
Votteler, Jörg
Wray, Victor
Schubert, Ulrich
2017-01-13T09:49:46Z
2017-01-13T09:49:46Z
2009-12-17
2015-09-04T08:31:10Z
BMC Structural Biology. 2009 Dec 17;9(1):74
http://dx.doi.org/10.1186/1472-6807-9-74
http://hdl.handle.net/10033/620696
Abstract Background The equine infection anemia virus (EIAV) p9 Gag protein contains the late (L-) domain required for efficient virus release of nascent virions from the cell membrane of infected cell. Results In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively) were chemically synthesized and used for structural analyses. Circular dichroism and 1H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this α-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein. Conclusions These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX) via the YPDL-motif to the site of virus budding, the counterpart of the YPXnL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101). The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.
Solution structure of the Equine Infectious Anemia Virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein
Journal Article
en
Sharma et al.2018-06-13T00:56:30ZAbstract
Background
The equine infection anemia virus (EIAV) p9 Gag protein contains the late (L-) domain required for efficient virus release of nascent virions from the cell membrane of infected cell.
Results
In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively) were chemically synthesized and used for structural analyses. Circular dichroism and 1H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this α-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein.
Conclusions
These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX) via the YPDL-motif to the site of virus budding, the counterpart of the YPXnL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101). The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.oai:repository.helmholtz-hzi.de:10033/5788812019-08-30T11:33:30Zcom_10033_6832com_10033_311308col_10033_620777col_10033_6833
Sun, Jibin
Lu, Xin
Rinas, Ursula
Zeng, An Ping
Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany.
2015-09-29T11:44:49Z
2015-09-29T11:44:49Z
2007
Metabolic peculiarities of Aspergillus niger disclosed by comparative metabolic genomics. 2007, 8 (9):R182 Genome Biol.
1474-760X
17784953
10.1186/gb-2007-8-9-r182
http://hdl.handle.net/10033/578881
Genome biology
Aspergillus niger is an important industrial microorganism for the production of both metabolites, such as citric acid, and proteins, such as fungal enzymes or heterologous proteins. Despite its extensive industrial applications, the genetic inventory of this fungus is only partially understood. The recently released genome sequence opens a new horizon for both scientific studies and biotechnological applications.
en
Aspergillus niger
Biotechnology
Citrate (si)-Synthase
Citric Acid
Fungal Proteins
Gene Expression Regulation, Fungal
Genes, Fungal
Genome
Genomics
Metabolism
Mitochondrial Proteins
Models, Biological
Models, Genetic
Models, Theoretical
Oxidoreductases
Phylogeny
Plant Proteins
Metabolic peculiarities of Aspergillus niger disclosed by comparative metabolic genomics.
Article2018-06-02T16:00:10ZAspergillus niger is an important industrial microorganism for the production of both metabolites, such as citric acid, and proteins, such as fungal enzymes or heterologous proteins. Despite its extensive industrial applications, the genetic inventory of this fungus is only partially understood. The recently released genome sequence opens a new horizon for both scientific studies and biotechnological applications.oai:repository.helmholtz-hzi.de:10033/5823392019-08-30T11:34:48Zcom_10033_6832col_10033_6833
Hebecker, Stefanie
Krausze, Joern
Hasenkampf, Tatjana
Schneider, Julia
Groenewold, Maike
Reichelt, Joachim
Jahn, Dieter
Heinz, Dirk W
Moser, Jürgen
Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany.
2015-11-18T15:59:03Z
2015-11-18T15:59:03Z
2015-08-25
Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine. 2015, 112 (34):10691-6 Proc. Natl. Acad. Sci. U.S.A.
1091-6490
26261323
10.1073/pnas.1511167112
http://hdl.handle.net/10033/582339
Proceedings of the National Academy of Sciences of the United States of America
The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.
en
Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine.
Article2018-06-13T09:22:17ZThe cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.oai:repository.helmholtz-hzi.de:10033/5831652019-08-30T11:36:05Zcom_10033_48156com_10033_6832col_10033_48157
Varga, John J
Barbier, Mariette
Mulet, Xavier
Bielecki, Piotr
Bartell, Jennifer A
Owings, Joshua P
Martinez-Ramos, Inmaculada
Hittle, Lauren E
Davis, Michael R
Damron, F Heath
Liechti, George W
Puchałka, Jacek
Dos Santos, Vitor A P Martins
Ernst, Robert K
Papin, Jason A
Albertí, Sebastian
Oliver, Antonio
Goldberg, Joanna B
HZI-Helmholtzzentrum für Infektionsforschung
2015-12-03T10:36:44Z
2015-12-03T10:36:44Z
2015
Genotypic and phenotypic analyses of a Pseudomonas aeruginosa chronic bronchiectasis isolate reveal differences from cystic fibrosis and laboratory strains. 2015, 16 (1):883 BMC Genomics
1471-2164
26519161
10.1186/s12864-015-2069-0
http://hdl.handle.net/10033/583165
BMC genomics
Pseudomonas aeruginosa is an environmentally ubiquitous Gram-negative bacterium and important opportunistic human pathogen, causing severe chronic respiratory infections in patients with underlying conditions such as cystic fibrosis (CF) or bronchiectasis. In order to identify mechanisms responsible for adaptation during bronchiectasis infections, a bronchiectasis isolate, PAHM4, was phenotypically and genotypically characterized.
en
Genotypic and phenotypic analyses of a Pseudomonas aeruginosa chronic bronchiectasis isolate reveal differences from cystic fibrosis and laboratory strains.
Article2018-06-13T14:07:06ZPseudomonas aeruginosa is an environmentally ubiquitous Gram-negative bacterium and important opportunistic human pathogen, causing severe chronic respiratory infections in patients with underlying conditions such as cystic fibrosis (CF) or bronchiectasis. In order to identify mechanisms responsible for adaptation during bronchiectasis infections, a bronchiectasis isolate, PAHM4, was phenotypically and genotypically characterized.oai:repository.helmholtz-hzi.de:10033/6040342019-08-30T11:37:44Zcom_10033_271853com_10033_6832col_10033_271872
Tarr, Alexander W
Khera, Tanvi
Hueging, Kathrin
Sheldon, Julie
Steinmann, Eike
Pietschmann, Thomas
Brown, Richard J P
TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover D-30625, Germany.
2016-03-30T14:15:08Z
2016-03-30T14:15:08Z
2015-07
Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design. 2015, 7 (7):3995-4046 Viruses
1999-4915
26193307
10.3390/v7072809
http://hdl.handle.net/10033/604034
Viruses
In the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with >90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design.
en
Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design.
Article2018-06-12T21:42:36ZIn the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with >90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design.oai:repository.helmholtz-hzi.de:10033/6036042019-08-30T11:37:44Zcom_10033_271853com_10033_6832col_10033_271872
Ignatev, Alexander
Bhargav, Saligram Prabhakar
Vahokoski, Juha
Kursula, Petri
Kursula, Inari
Helmholtz Centre for Infection Research, University of Hamburg, and German Electron Synchrotron (DESY), Hamburg, Germany.
2016-03-24T12:01:45Z
2016-03-24T12:01:45Z
2012
The lasso segment is required for functional dimerization of the Plasmodium formin 1 FH2 domain. 2012, 7 (3):e33586 PLoS ONE
1932-6203
22428073
10.1371/journal.pone.0033586
http://hdl.handle.net/10033/603604
PloS one
Apicomplexan parasites, such as the malaria-causing Plasmodium species, utilize a unique way of locomotion and host cell invasion. This substrate-dependent gliding motility requires rapid cycling of actin between the monomeric state and very short, unbranched filaments. Despite the crucial role of actin polymerization for the survival of the malaria parasite, the majority of Plasmodium cellular actin is present in the monomeric form. Plasmodium lacks most of the canonical actin nucleators, and formins are essentially the only candidates for this function in all Apicomplexa. The malaria parasite has two formins, containing conserved formin homology (FH) 2 and rudimentary FH1 domains. Here, we show that Plasmodium falciparum formin 1 associates with and nucleates both mammalian and Plasmodium actin filaments. Although Plasmodium profilin alone sequesters actin monomers, thus inhibiting polymerization, its monomer-sequestering activity does not compete with the nucleating activity of formin 1 at an equimolar profilin-actin ratio. We have determined solution structures of P. falciparum formin 1 FH2 domain both in the presence and absence of the lasso segment and the FH1 domain, and show that the lasso is required for the assembly of functional dimers.
en
info:eu-repo/grantAgreement/EC/FP7/226716
openAccess
Actins
Amino Acid Sequence
Circular Dichroism
Cloning, Molecular
Dimerization
Fetal Proteins
Locomotion
Microfilament Proteins
Models, Molecular
Molecular Sequence Data
Nuclear Proteins
Plasmodium falciparum
Protein Structure, Tertiary
Scattering, Small Angle
The lasso segment is required for functional dimerization of the Plasmodium formin 1 FH2 domain.
Article2018-06-12T23:14:16ZApicomplexan parasites, such as the malaria-causing Plasmodium species, utilize a unique way of locomotion and host cell invasion. This substrate-dependent gliding motility requires rapid cycling of actin between the monomeric state and very short, unbranched filaments. Despite the crucial role of actin polymerization for the survival of the malaria parasite, the majority of Plasmodium cellular actin is present in the monomeric form. Plasmodium lacks most of the canonical actin nucleators, and formins are essentially the only candidates for this function in all Apicomplexa. The malaria parasite has two formins, containing conserved formin homology (FH) 2 and rudimentary FH1 domains. Here, we show that Plasmodium falciparum formin 1 associates with and nucleates both mammalian and Plasmodium actin filaments. Although Plasmodium profilin alone sequesters actin monomers, thus inhibiting polymerization, its monomer-sequestering activity does not compete with the nucleating activity of formin 1 at an equimolar profilin-actin ratio. We have determined solution structures of P. falciparum formin 1 FH2 domain both in the presence and absence of the lasso segment and the FH1 domain, and show that the lasso is required for the assembly of functional dimers.oai:repository.helmholtz-hzi.de:10033/6159852019-08-30T11:31:23Zcom_10033_271853com_10033_6832col_10033_271872
Raasakka, Arne
Myllykoski, Matti
Laulumaa, Saara
Lehtimäki, Mari
Härtlein, Michael
Moulin, Martine
Kursula, Inari
Kursula, Petri
CSSB, Centre for Structural Systems Biology, Notekestr. 85, 22607 Hamburg, Germany.
2016-07-12T14:06:57Z
2016-07-12T14:06:57Z
2015
Determinants of ligand binding and catalytic activity in the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase. 2015, 5:16520 Sci Rep
2045-2322
26563764
10.1038/srep16520
http://hdl.handle.net/10033/615985
Scientific reports
2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enzyme highly abundant in the central nervous system myelin of terrestrial vertebrates. The catalytic domain of CNPase belongs to the 2H phosphoesterase superfamily and catalyzes the hydrolysis of nucleoside 2',3'-cyclic monophosphates to nucleoside 2'-monophosphates. The detailed reaction mechanism and the essential catalytic amino acids involved have been described earlier, but the roles of many amino acids in the vicinity of the active site have remained unknown. Here, several CNPase catalytic domain mutants were studied using enzyme kinetics assays, thermal stability experiments, and X-ray crystallography. Additionally, the crystal structure of a perdeuterated CNPase catalytic domain was refined at atomic resolution to obtain a detailed view of the active site and the catalytic mechanism. The results specify determinants of ligand binding and novel essential residues required for CNPase catalysis. For example, the aromatic side chains of Phe235 and Tyr168 are crucial for substrate binding, and Arg307 may affect active site electrostatics and regulate loop dynamics. The β5-α7 loop, unique for CNPase in the 2H phosphoesterase family, appears to have various functions in the CNPase reaction mechanism, from coordinating the nucleophilic water molecule to providing a binding pocket for the product and being involved in product release.
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Determinants of ligand binding and catalytic activity in the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase.
Article2018-06-12T20:06:15Z2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enzyme highly abundant in the central nervous system myelin of terrestrial vertebrates. The catalytic domain of CNPase belongs to the 2H phosphoesterase superfamily and catalyzes the hydrolysis of nucleoside 2',3'-cyclic monophosphates to nucleoside 2'-monophosphates. The detailed reaction mechanism and the essential catalytic amino acids involved have been described earlier, but the roles of many amino acids in the vicinity of the active site have remained unknown. Here, several CNPase catalytic domain mutants were studied using enzyme kinetics assays, thermal stability experiments, and X-ray crystallography. Additionally, the crystal structure of a perdeuterated CNPase catalytic domain was refined at atomic resolution to obtain a detailed view of the active site and the catalytic mechanism. The results specify determinants of ligand binding and novel essential residues required for CNPase catalysis. For example, the aromatic side chains of Phe235 and Tyr168 are crucial for substrate binding, and Arg307 may affect active site electrostatics and regulate loop dynamics. The β5-α7 loop, unique for CNPase in the 2H phosphoesterase family, appears to have various functions in the CNPase reaction mechanism, from coordinating the nucleophilic water molecule to providing a binding pocket for the product and being involved in product release.oai:repository.helmholtz-hzi.de:10033/6210492019-08-30T11:35:39Zcom_10033_6832col_10033_6833
Jäger, Jens
Marwitz, Sebastian
Tiefenau, Jana
Rasch, Janine
Shevchuk, Olga
Kugler, Christian
Goldmann, Torsten
Steinert, Michael
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
2017-08-08T11:25:42Z
2017-08-08T11:25:42Z
2014-01
Human lung tissue explants reveal novel interactions during Legionella pneumophila infections. 2014, 82 (1):275-85 Infect. Immun.
1098-5522
24166955
10.1128/IAI.00703-13
http://hdl.handle.net/10033/621049
Infection and immunity
Histological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model for Legionella pneumophila infection comprising living human lung tissue. We stimulated lung explants with L. pneumophila strains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion of L. pneumophila to the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA(-) strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context of L. pneumophila infections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations.
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Adhesins, Bacterial
Bacterial Adhesion
Gene Expression Regulation, Bacterial
Humans
Interferon-gamma
Legionella pneumophila
Legionnaires' Disease
Lung
Macrophages, Alveolar
Models, Biological
RNA, Bacterial
Transcriptome
Human lung tissue explants reveal novel interactions during Legionella pneumophila infections.
Article2018-06-13T02:28:24ZHistological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model for Legionella pneumophila infection comprising living human lung tissue. We stimulated lung explants with L. pneumophila strains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion of L. pneumophila to the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA(-) strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context of L. pneumophila infections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations.oai:repository.helmholtz-hzi.de:10033/6211142019-08-30T11:37:24Zcom_10033_6832col_10033_6833
Schniederjans, Monika
Koska, Michal
Häussler, Susanne
Helmholtz Centre for infection researchGmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
2017-09-22T08:16:17Z
2017-09-22T08:16:17Z
2017-09-05
Transcriptional and mutational profiling of an aminoglycoside resistant Pseudomonas aeruginosa small colony variant. 2017 Antimicrob. Agents Chemother.
1098-6596
28874369
10.1128/AAC.01178-17
http://hdl.handle.net/10033/621114
Antimicrobial agents and chemotherapy
Pseudomonas aeruginosa is a major causative agent of both acute and chronic infections. Although aminoglycoside antibiotics are very potent drugs to fight such infections, antibiotic failure is steadily increasing mainly due to increasing resistance of the bacteria. Many molecular mechanisms that determine resistance such as acquisition of genes encoding for aminoglycoside-inactivating enzymes or overexpression of efflux pumps have been elucidated. However, there are additional, less-well described mechanisms of aminoglycoside resistance. In this study we have profiled a clinical tobramycin resistant P. aeruginosa strain that exhibited a small colony variant (SCV) phenotype. Both, the resistance and the colony morphology phenotypes were lost upon passaging the isolate under rich medium conditions. Transcriptional and mutational profiling revealed that the SCV harbored activating mutations in the two two-component systems AmgRS and PmrAB. Introduction of these mutations singularly into the type strain PA14 conferred tobramycin and colistin resistance, respectively. However, their combined introduction had an additive effect on the tobramycin resistance phenotype. Activation of the AmgRS system slightly reduced the colony size of the PA14 wild-type, whereas the simultaneous overexpression of gacA, the response regulator of the GacSA two component system, further reduced colony size. In conclusion, we uncovered combinatorial influences of two-component systems on clinically relevant phenotypes, such as resistance and the expression of the SCV phenotype. Our results clearly demonstrate that combined activation of P. aeruginosa two-component systems exhibit pleiotropic effects with unforeseen consequences.
en
info:eu-repo/grantAgreement/EC/FP7/ 260276
embargoedAccess
http://creativecommons.org/licenses/by-nc-sa/4.0/
Transcriptional and mutational profiling of an aminoglycoside resistant Pseudomonas aeruginosa small colony variant.
Article2018-03-05T00:00:00ZPseudomonas aeruginosa is a major causative agent of both acute and chronic infections. Although aminoglycoside antibiotics are very potent drugs to fight such infections, antibiotic failure is steadily increasing mainly due to increasing resistance of the bacteria. Many molecular mechanisms that determine resistance such as acquisition of genes encoding for aminoglycoside-inactivating enzymes or overexpression of efflux pumps have been elucidated. However, there are additional, less-well described mechanisms of aminoglycoside resistance. In this study we have profiled a clinical tobramycin resistant P. aeruginosa strain that exhibited a small colony variant (SCV) phenotype. Both, the resistance and the colony morphology phenotypes were lost upon passaging the isolate under rich medium conditions. Transcriptional and mutational profiling revealed that the SCV harbored activating mutations in the two two-component systems AmgRS and PmrAB. Introduction of these mutations singularly into the type strain PA14 conferred tobramycin and colistin resistance, respectively. However, their combined introduction had an additive effect on the tobramycin resistance phenotype. Activation of the AmgRS system slightly reduced the colony size of the PA14 wild-type, whereas the simultaneous overexpression of gacA, the response regulator of the GacSA two component system, further reduced colony size. In conclusion, we uncovered combinatorial influences of two-component systems on clinically relevant phenotypes, such as resistance and the expression of the SCV phenotype. Our results clearly demonstrate that combined activation of P. aeruginosa two-component systems exhibit pleiotropic effects with unforeseen consequences.