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Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/41701
Title: Insights into protein folding mechanisms from large scale analysis of mutational effects.
Authors: Naganathan, Athi N.; Muñoz van den Eynde, Víctor
Keywords: Protein kinetics
Protein mutations
Protein folding
Protein Stability
Protein Structure
Free energy relationships
Perturbation analysis
Issue Date: 11-May-2010
Publisher: National Academy of Sciences (U.S.)
Citation: Proceedings of the National Academy of Sciences of the USA, 107(19): 8611-8616
Abstract: Protein folding mechanisms are probed experimentally using single-point mutant perturbations. The relative effects on the folding (phi-values) and unfolding (1 - phi) rates are used to infer the detailed structure of the transition-state ensemble (TSE). Here we analyze kinetic data on > 800 mutations carried out for 24 proteins with simple kinetic behavior. We find two surprising results: (i) all mutant effects are described by the equation: DeltaDeltaG(double dagger)(unfold)=0.76DeltaDeltaG(eq) +/- 1.8kJ/mol. Therefore all data are consistent with a single phi-value (0.24) with accuracy comparable to experimental precision, suggesting that the structural information in conventional phi-values is low. (ii) phi-values change with stability, increasing in mean value and spread from native to unfolding conditions, and thus cannot be interpreted without proper normalization. We eliminate stability effects calculating the phi-values at the mutant denaturation midpoints; i.e., conditions of zero stability (phi(0)). We then show that the intrinsic variability is phi(0) = 0.36 +/- 0.11, being somewhat larger for beta-sheet-rich proteins than for alpha-helical proteins. Importantly, we discover that phi(0)-values are proportional to how many of the residues surrounding the mutated site are local in sequence. High phi(0)-values correspond to protein surface sites, which have few nonlocal neighbors, whereas core residues with many tertiary interactions produce the lowest phi(0)-values. These results suggest a general mechanism in which the TSE at zero stability is a broad conformational ensemble stabilized by local interactions and without specific tertiary interactions, reconciling phi-values with many other empirical observations.
Description: This article contains supporting information online at www.pnas.org/cgi/content/full/1000988107/DCSupplemental and http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889297/bin/supp_107_19_8611__index.html
Publisher version (URL): http://www.pnas.org/content/107/19/8611.long
URI: http://hdl.handle.net/10261/41701
DOI: 10.1073/pnas.1000988107
ISSN: 1091-6490
References: PMC2889297
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