Recognition between a short unstructured peptide and a partially folded fragment leads to the thioredoxin fold sharing native-like dynamics

Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina.
Proteins Structure Function and Bioinformatics (Impact Factor: 2.63). 05/2012; 80(5):1448-64. DOI: 10.1002/prot.24043
Source: PubMed

ABSTRACT Thioredoxins (TRXs) constitute attractive α/β scaffolds for investigating molecular recognition. The interaction between the recombinant fragment spanning the sequence 1-93 of full-length TRX (TRX1-93) and the synthetic peptide comprising residues 94-108 (TRX94-108), plus a C-terminal tyrosine tag (the numbering scheme used in entry pdb 2TRX is used throughout the article, two complementary moieties of E. coli TRX, brings about the consolidation of a native-like complex. Despite its reduced thermodynamic stability, this complex is able to acquire fine structural features remarkably similar to those characteristic of full-length TRX, namely, hydrodynamic behavior, assessed by diffusion-ordered spectroscopy (DOSY)-NMR; the pattern of secondary structure, as revealed by three-bond HNHα coupling constants and secondary shifts for Hα/CO/Cα/Cβ; native-like tertiary structural signatures revealed by near-UV circular dichroism (CD) spectroscopy. The complex exhibits a relaxation behavior compatible with that expected for a native-like structure. However, heteronuclear nuclear Overhauser effect (NOE)s reveal an enhanced dynamics for the complex by comparison with full-length TRX. Furthermore, higher R(2) values for residues 43-50 and 74-89 would likely result from an exchange process modulated by the peptide at the interface region. The slow kinetics of the consolidation reaction was followed by CD and real-time NMR. Equilibrium titration experiments by NMR yield a K(D) value of 1.4 ± 1.0 μM and a second low-affinity (>150 μM) binding event in the vicinity of the active site. Molecular dynamics simulations of both the isolated fragment TRX1-93 and the complex suggest the destabilization of α2 and α3 helical elements and the persistence of β-structure in the absence of TRX94-108. Altogether, structural and dynamic evidence presented herein points to the key role played by the C-terminal helix in establishing the overall fold. This critical switch module endows reduced TRX with the ability to act as a cooperative folding unit.

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Available from: Javier Santos, Sep 27, 2015
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    • "However, heteronuclear nuclear Overhauser effect (hnNOE) reveals an enhanced dynamics. The higher R2 values for residues 43– 50 and 74–89 are compatible with an exchange process modulated by the peptide at the interface region [154]. In "
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    ABSTRACT: Thioredoxin (TRX) catalyzes redox reactions via the reversible oxidation of the conserved active center CGPC and it is involved in multiple biological processes, some of them linked to redox activity while others not. TRX is a globular, thermodynamically stable and monomeric alpha/beta protein with a structure characterized by a central beta-sheet surrounded by alpha-helices. In this review we discuss central aspects of folding, dynamics and function of Escherichia coli TRX (EcTRX), pointing to the characterization of the full-length protein and of relevant fragments. In addition, we focus on the critical role that the C-terminal alpha-helical element plays in a late event in the consolidation of the overall EcTRX fold. Furthermore, we address the characterization of internal molecular motions by NMR and molecular dynamics simulation techniques. Finally, we review important aspects of the relationship among structure, dynamics and enzymatic function of this key redox protein.
    Protein and Peptide Letters 07/2015; 22(9). DOI:10.2174/0929866522666150707114309 · 1.07 Impact Factor
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    • "Experiments between different couples of EcTRX fragments point to the plasticity of the core of this protein [35] [36] [37] [38] [39] [40] and to the relevance of C-terminal α-helix (CTH) on the consolidation of the native state [41] [42]. Experiments involving fragment TRX1–93 showed that the absence of the CTH (residues 94–108) results in the stabilization of a premolten globule-like state, with only a residual secondary structure and without signatures of a persistent tertiary structure. "
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    ABSTRACT: In this work, the unfolding mechanism of oxidized Escherichia coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against global unfolding while leaving the native structure unchanged. Global analysis of the unfolding kinetics of all variants revealed a linear unfolding route with a high-energy on-pathway intermediate state flanked by two transition state ensembles TSE1 and TSE2. The experiments show that CTH is mainly unfolded in TSE1 and the intermediate and becomes structured in TSE2. Structure-based molecular dynamics are in agreement with these experiments and provide protein-wide structural information on transient states. In our model, EcTRX folding starts with structure formation in the β-sheet, while the protein helices coalesce later. As a whole, our results indicate that the CTH is a critical module in the folding process, restraining a heterogeneous intermediate ensemble into a biologically active native state and providing the native protein with thermodynamic and kinetic stability.
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    ABSTRACT: NMR spectroscopy is one of the few biophysical methods that can provide atomic-level insight into the conformation of partially folded states and/or intermediates present along the protein folding pathway. Such studies are important not only within the context of the protein folding problem, but also to push forward the technique, due to the challenging nature of the systems studied. In fact, new NMR methods have been created, and applied, in an attempt to characterize the conformational features of the states along the folding pathway. Describing the structures along the folding landscape is of key importance to comprehend the folding reaction, design new proteins and to understand how several polypeptide chains are implicated in pathogenic amyloid states. The last advances in several approaches, which use NMR: (i) to monitor the protein folding pathway and/or, (ii) to characterize the structure of the intermediate states in such reaction are reviewed in this work.
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