Production, biophysical characterization and crystallization of Pseudomonas putida GraA and its complexes with GraT and the graTA operator.

TitleProduction, biophysical characterization and crystallization of Pseudomonas putida GraA and its complexes with GraT and the graTA operator.
Publication TypeJournal Article
Year of Publication2017
AuthorsTalavera, A., H. Tamman, A. Ainelo, S. Hadǽi, A. Garcia-Pino, R. Hõrak, A. Konijnenberg, and R. Loris
JournalActa Crystallogr F Struct Biol Commun
Volume73
IssuePt 8
Pagination455-462
Date Published2017 Aug 01
ISSN2053-230X
KeywordsAmino Acid Sequence, Antitoxins, Bacterial Proteins, Bacterial Toxins, Cloning, Molecular, Crystallization, Crystallography, X-Ray, Escherichia coli, Gene Expression, Gene Expression Regulation, Bacterial, Genetic Vectors, Operon, Protein Binding, Protein Multimerization, Pseudomonas putida, Recombinant Fusion Proteins, X-Ray Diffraction
Abstract

The graTA operon from Pseudomonas putida encodes a toxin-antitoxin module with an unusually moderate toxin. Here, the production, SAXS analysis and crystallization of the antitoxin GraA, the GraTA complex and the complex of GraA with a 33 bp operator fragment are reported. GraA forms a homodimer in solution and crystallizes in space group P2, with unit-cell parameters a = 66.9, b = 48.9, c = 62.7 Å, β = 92.6°. The crystals are likely to contain two GraA dimers in the asymmetric unit and diffract to 1.9 Å resolution. The GraTA complex forms a heterotetramer in solution. Crystals of the GraTA complex diffracted to 2.2 Å resolution and are most likely to contain a single heterotetrameric GraTA complex in the asymmetric unit. They belong to space group P4 or P4, with unit-cell parameters a = b = 56.0, c = 128.2 Å. The GraA-operator complex consists of a 33 bp operator region that binds two GraA dimers. It crystallizes in space group P3 or P3, with unit-cell parameters a = b = 105.6, c = 149.9 Å. These crystals diffract to 3.8 Å resolution.

DOI10.1107/S2053230X17009438
Alternate JournalActa Crystallogr F Struct Biol Commun
PubMed ID28777088
Grant ListG.0135.15N / / Fonds Wetenschappelijk Onderzoek /
G0C1213N / / Fonds Wetenschappelijk Onderzoek /
UABR/11/012 / / The Hercules Foundation /
IUT20-19 / / Estonian Research Council /
PUT1351 / / Estonian Research Council /
OZR2232 / / Vrije Universiteit Brussel /
SPR13 / / Vrije Universiteit Brussel /
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