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Sci Rep. 2017 Feb 9;7:42343. doi: 10.1038/srep42343.

Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants.

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Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas, CONICET, Junín 956, C1113AAD, Buenos Aires, Argentina.
Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica and Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, Uruguay.
Department of Integrative Biology, IGBMC, CNRS, INSERM, Université de Strasbourg, Illkirch, France.
Instituto de Biología Molecular y Celular de Rosario (IBR), Rosario, Santa Fe, Argentina.


Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.

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