Detection and Structure Determination of an Equilibrium Unfolding Intermediate of Rd-apocytochrome b562: Native Fold with Non-native Hydrophobic Interactions

Feng, Hanqiao; Vu, Ngoc-Diep; Bai, Yawen

doi:10.1016/j.jmb.2004.08.099

Cited by 27 publications

(19 citation statements)

References 58 publications

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“…This templating occurs both on the route up to the TSE (20) and on the descent down (34,35). The incremental buildup of secondary and tertiary structure mostly produces native-or unfolded-like regions, as suggested by the frequent observation of ψ = 0 or 1 and the cooperative pattern of hydrogen exchange (HX) protection factors within secondary structures (36)(37)(38)(39). Folding steps can involve both local and nonlocal contacts and H-bonds even during the early stages of folding.…”

Section: Discussionmentioning

confidence: 99%

Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar

Baxa

Adhikari

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Experimental and computational folding studies of Proteins L & G and NuG2 typically find that sequence differences determine which of the two hairpins is formed in the transition state ensemble (TSE). However, our recent work on Protein L finds that its TSE contains both hairpins, compelling a reassessment of the influence of sequence on the folding behavior of the other two homologs. We characterize the TSEs for Protein G and NuG2b, a triple mutant of NuG2, using ψ analysis, a method for identifying contacts in the TSE. All three homologs are found to share a common and near-native TSE topology with interactions between all four strands. However, the helical content varies in the TSE, being largely absent in Proteins G & L but partially present in NuG2b. The variability likely arises from competing propensities for the formation of nonnative β turns in the naturally occurring proteins, as observed in our TerItFix folding algorithm. All-atom folding simulations of NuG2b recapitulate the observed TSEs with four strands for 5 of 27 transition paths [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517–520]. Our data support the view that homologous proteins have similar folding mechanisms, even when nonnative interactions are present in the transition state. These findings emphasize the ongoing challenge of accurately characterizing and predicting TSEs, even for relatively simple proteins.

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Section: Discussionmentioning

confidence: 99%

Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar

Baxa

Adhikari

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Native state hydrogen exchange (NSHX) studies have shown that subglobal unfolding events occur in many proteins (Bai and Englander 1996;Chamberlain et al 1996;Feng et al 2004a). These openings represent the unfolding of individual or groups of SecStr elements and highlight that protein folding is not completely cooperative even for proteins with apparent two-state kinetics.…”

Section: Steps In Protein Foldingmentioning

confidence: 99%

Kinetic barriers and the role of topology in protein and RNA folding

Sosnick

2008

Protein Science

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This review compares the folding behavior of proteins and RNAs. Topics covered include the role of topology in the determination of folding rates, major folding events including collapse, properties of denatured states, pathway heterogeneity, and the influence of the mode of initiation on the folding pathway.Keywords: protein structure/folding; RNA folding Proteins and RNAs must fold into specific structures in order to carry out their functions within the cell. For both biomolecules, the stability and specificity of the native fold results from the burial of hydrophobic and aromatic side groups, and the formation of hydrogen bonds. Nevertheless, their folding behavior differs in many important ways (Thirumalai and Hyeon 2005)( Table 1). Proteins have a higher folding cooperativity with secondary structure (SecStr) and collapse occurring in the same kinetic event. In contrast, RNA SecStr typically forms at low ionic conditions with collapse and tertiary structure (TertStr) formation occurring only upon the addition of counterions that shield the repulsion of the negatively charged RNA phosphodiester backbone. Specific Mg 2+ binding often helps assemble the preformed RNA SecStrs into tertiary architectures (Cate et al. 1997;Batey et al. 1999;Klein et al. 2004). As a result, the protein folding landscape has fewer stable intermediates than the RNA folding landscape (Thirumalai and Woodson 1996;Treiber et al. 1998;Thirumalai et al. 2001;Treiber and Williamson 2001;Das et al. 2003;Baird et al. 2007). Folding rates and chain topologyWhen interpreting data and identifying the critical folding events, one must distinguish between the intrinsic (unavoidable) kinetic barriers from the optional barriers, which arise on some pathways but not on others. Proteins and RNAs can fold directly to the native state or be trapped by kinetic barriers related to undoing errors formed early in the pathway (Bryngelson and Wolynes 1989;Abkevich et al. 1994a;Sosnick et al. 1994;Guo and Thirumalai 1995;Thirumalai and Woodson 1996;Pan and Sosnick 1997;Pan et al. , 1999bTreiber and Williamson 1999;Russell et al. 2002). The nature of the errors include premature collapse and nonnative secondary structure formation. These optional barriers obscure the intrinsic barriers, those unavoidable steps that determine folding rates in the absence of error correction. Protein foldingThe error-free folding of proteins had been proposed to be a nucleation process where the chain attains a coarse

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“…Figure 6(a)-(g) illustrate the chevron plots for the mutants. To obtain the global unfolding free energy for each mutant, we fitted the region near the bottom of the chevron plot to the following equation: (5) where and are the folding and unfolding rate constants in the absence of denaturant; and m f and m u are the parameters describing the slope of folding and unfolding limbs of chevron plot. We chose to calculate the Φ values at 1 M urea since most of the folding rates of mutants were measured directly at 1 M urea.…”

Section: φ-Value Analysismentioning

confidence: 99%

The Folding Pathway of T4 Lysozyme: The High-resolution Structure and Folding of a Hidden Intermediate

Kato

Feng

Bai

2007

Journal of Molecular Biology

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Folding intermediates have been detected and characterized for many proteins. However, their structures at atomic resolution have only been determined for two small single domain proteins: Rdapocytochrome b 562 and engrailed homeo domain. T4 lysozyme has two easily distinguishable but energetically coupled domains: the N-and C-terminal domains. An early native-state hydrogen exchange experiment identified an intermediate with the C-terminal domain folded and the Nterminal domain unfolded. We have used a native-state hydrogen exchange-directed protein engineering approach to populate this intermediate and demonstrated that it is on the folding pathway and exists after the rate-limiting step. Here, we determined its high-resolution structure and the backbone dynamics by multi-dimensional NMR methods. We also characterized the folding behavior of the intermediate using stopped-flow fluorescence, protein engineering, and native-state hydrogen exchange. Unlike the folding intermediates of the two single-domain proteins, which have many nonnative side chain interactions, the structure of the hidden folding intermediate of T4 lysozyme is largely native-like. It folds like many small single domain proteins. These results have implications for understanding the folding mechanism and evolution of multi-domain proteins.

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Detection and Structure Determination of an Equilibrium Unfolding Intermediate of Rd-apocytochrome b562: Native Fold with Non-native Hydrophobic Interactions

Cited by 27 publications

References 58 publications

Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar

Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar

Kinetic barriers and the role of topology in protein and RNA folding

The Folding Pathway of T4 Lysozyme: The High-resolution Structure and Folding of a Hidden Intermediate

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