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Amino acid residues important for folding of thioredoxin are revealed only by study of the physiologically relevant reduced form of the protein.

ABSTRACT: Thioredoxin-1 from Escherichia coli has frequently been used as a model substrate in protein folding studies. However, for reasons of convenience, these studies have focused largely on oxidized thioredoxin and not on reduced thioredoxin, the more physiologically relevant species. Here we describe the first extensive characterization of the refolding kinetics and conformational thermodynamics of reduced thioredoxin. We have previously described a genetic screen that yielded mutant thioredoxin proteins that fold more slowly in both the oxidized and reduced forms. In this study, we apply our more detailed analysis of reduced thioredoxin folding to a larger number of folding mutants that includes those obtained from continuation of the genetic screen. We have identified mutant proteins that display folding defects specifically in the reduced state but not the oxidized state. Some of these substitutions represent unusual folding mutants in that they result in semiconservative substitutions at solvent-exposed positions in the folded conformation and do not appear to affect the conformational stability of the protein. Further, the genetic selection yields mutants at only a limited number of sites, pointing to perhaps the most critical amino acids in the folding pathway and underscoring, in particular, the role of the carboxy-terminal amino acids in the folding of thioredoxin. Our results demonstrate the importance of studying the physiologically relevant folding species.

SUBMITTER: Huber D

PROVIDER: S-EPMC2953564 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Amino acid residues important for folding of thioredoxin are revealed only by study of the physiologically relevant reduced form of the protein.

Huber Damon D Chaffotte Alain A Eser Markus M Planson Anne-Gaëlle AG Beckwith Jon J

Biochemistry 20101001 41

Thioredoxin-1 from Escherichia coli has frequently been used as a model substrate in protein folding studies. However, for reasons of convenience, these studies have focused largely on oxidized thioredoxin and not on reduced thioredoxin, the more physiologically relevant species. Here we describe the first extensive characterization of the refolding kinetics and conformational thermodynamics of reduced thioredoxin. We have previously described a genetic screen that yielded mutant thioredoxin pro ...[more]

PMID: 20873718

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Project description:BackgroundWell-performing automated protein function recognition approaches usually comprise several complementary techniques. Beside constructing better consensus, their predictive power can be improved by either adding or refining independent modules that explore orthogonal features of proteins. In this work, we demonstrated how the exploration of global atomic distributions can be used to indicate functionally important residues.ResultsUsing a set of carefully selected globular proteins, we parametrized continuous probability density functions describing preferred central distances of individual protein atoms. Relative preferred burials were estimated using mixture models of radial density functions dependent on the amino acid composition of a protein under consideration. The unexpectedness of extraordinary locations of atoms was evaluated in the information-theoretic manner and used directly for the identification of key amino acids. In the validation study, we tested capabilities of a tool built upon our approach, called SurpResi, by searching for binding sites interacting with ligands. The tool indicated multiple candidate sites achieving success rates comparable to several geometric methods. We also showed that the unexpectedness is a property of regions involved in protein-protein interactions, and thus can be used for the ranking of protein docking predictions. The computational approach implemented in this work is freely available via a Web interface at http://www.bioinformatics.org/surpresi.ConclusionsProbabilistic analysis of atomic central distances in globular proteins is capable of capturing distinct orientational preferences of amino acids as resulting from different sizes, charges and hydrophobic characters of their side chains. When idealized spatial preferences can be inferred from the sole amino acid composition of a protein, residues located in hydrophobically unfavorable environments can be easily detected. Such residues turn out to be often directly involved in binding ligands or interfacing with other proteins.

Dataset Information

Amino acid residues important for folding of thioredoxin are revealed only by study of the physiologically relevant reduced form of the protein.

Publications

Amino acid residues important for folding of thioredoxin are revealed only by study of the physiologically relevant reduced form of the protein.

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