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Title: Combining site-specific mutagenesis and seeding as a strategy to crystallize ‘difficult’ proteins: the case of Staphylococcus aureus thioredoxin

Abstract

S. aureus thioredoxin was crystallized using a combination of seeding and site-specific mutagenesis. The P31T mutant of Staphylococcus aureus thioredoxin crystallizes spontaneously in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 41.7, b = 49.5, c = 55.6 Å. The crystals diffract to 2.2 Å resolution. Isomorphous crystals of wild-type thioredoxin as well as of other point mutants only grow when seeded with the P31T mutant. These results suggest seeding as a valuable tool complementing surface engineering for proteins that are hard to crystallize.

Authors:
 [1]; ;  [1]; ;  [2];  [3];  [1];  [3]
  1. Brussels Center for Redox Biology, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB) at the Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Brussel (Belgium)
  2. Architecture et Fonction des Macromolecules Biologiques, UMR 6098 CNRS and Universites d’Aix-Marseille I et II, Campus de Luminy, 163 Avenue de Luminy 13288 Marseille CEDEX 9 (France)
  3. Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB) at the Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Brussel (Belgium)
Publication Date:
OSTI Identifier:
22356421
Resource Type:
Journal Article
Journal Name:
Acta Crystallographica. Section F
Additional Journal Information:
Journal Volume: 62; Journal Issue: Pt 12; Other Information: PMCID: PMC2225371; PMID: 17142910; PUBLISHER-ID: ll5093; OAI: oai:pubmedcentral.nih.gov:2225371; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 1744-3091
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALS; MUTANTS; RESOLUTION; SPACE GROUPS; STAPHYLOCOCCUS; SURFACES

Citation Formats

Roos, Goedele, Algemene Chemie, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Brosens, Elke, Wahni, Khadija, Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Desmyter, Aline, Spinelli, Silvia, Wyns, Lode, Messens, Joris, Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Loris, Remy, and Brussels Center for Redox Biology, Vlaams Interuniversitair Instituut voor Biotechnologie. Combining site-specific mutagenesis and seeding as a strategy to crystallize ‘difficult’ proteins: the case of Staphylococcus aureus thioredoxin. United Kingdom: N. p., 2006. Web. doi:10.1107/S1744309106047075.
Roos, Goedele, Algemene Chemie, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Brosens, Elke, Wahni, Khadija, Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Desmyter, Aline, Spinelli, Silvia, Wyns, Lode, Messens, Joris, Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Loris, Remy, & Brussels Center for Redox Biology, Vlaams Interuniversitair Instituut voor Biotechnologie. Combining site-specific mutagenesis and seeding as a strategy to crystallize ‘difficult’ proteins: the case of Staphylococcus aureus thioredoxin. United Kingdom. https://doi.org/10.1107/S1744309106047075
Roos, Goedele, Algemene Chemie, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Brosens, Elke, Wahni, Khadija, Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Desmyter, Aline, Spinelli, Silvia, Wyns, Lode, Messens, Joris, Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Loris, Remy, and Brussels Center for Redox Biology, Vlaams Interuniversitair Instituut voor Biotechnologie. 2006. "Combining site-specific mutagenesis and seeding as a strategy to crystallize ‘difficult’ proteins: the case of Staphylococcus aureus thioredoxin". United Kingdom. https://doi.org/10.1107/S1744309106047075.
@article{osti_22356421,
title = {Combining site-specific mutagenesis and seeding as a strategy to crystallize ‘difficult’ proteins: the case of Staphylococcus aureus thioredoxin},
author = {Roos, Goedele and Algemene Chemie, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel and Brosens, Elke and Wahni, Khadija and Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie and Desmyter, Aline and Spinelli, Silvia and Wyns, Lode and Messens, Joris and Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie and Loris, Remy and Brussels Center for Redox Biology, Vlaams Interuniversitair Instituut voor Biotechnologie},
abstractNote = {S. aureus thioredoxin was crystallized using a combination of seeding and site-specific mutagenesis. The P31T mutant of Staphylococcus aureus thioredoxin crystallizes spontaneously in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 41.7, b = 49.5, c = 55.6 Å. The crystals diffract to 2.2 Å resolution. Isomorphous crystals of wild-type thioredoxin as well as of other point mutants only grow when seeded with the P31T mutant. These results suggest seeding as a valuable tool complementing surface engineering for proteins that are hard to crystallize.},
doi = {10.1107/S1744309106047075},
url = {https://www.osti.gov/biblio/22356421}, journal = {Acta Crystallographica. Section F},
issn = {1744-3091},
number = Pt 12,
volume = 62,
place = {United Kingdom},
year = {Fri Dec 01 00:00:00 EST 2006},
month = {Fri Dec 01 00:00:00 EST 2006}
}