High-Resolution Mapping of the Folding Transition State of a WW Domain

JOURNAL OF MOLECULAR BIOLOGY

卷:428

Article

Dave, Kapil¹  Jager, Marcus^2,3  Houbi Nguyen¹  Kelly, Jeffery W.^2,3  Gruebele, Martin^1,4,5 

[1] Univ Illinois Urbana Champaign, Ctr Biophys & Computat Biol, Urbana, IL 61801 USA

[2] Scripps Res Inst, Dept Chem, 10550 North Torrey Pines Rd BCC255, La Jolla, CA 92037 USA

[3] Scripps Res Inst, Skaggs Inst Chem Biol, 10550 North Torrey Pines Rd BCC255, La Jolla, CA 92037 USA

[4] Univ Illinois Urbana Champaign, Dept Phys, Urbana, IL 61801 USA

[5] Univ Illinois Urbana Champaign, Dept Chem, Urbana, IL 61801 USA

关键词: protein folding;    WW domain;    Phi-value analysis;    folding transition state;    laser T-jump;   

DOI  :  10.1016/j.jmb.2016.02.008

来源: Elsevier

【 摘 要 】

Fast-folding WW domains are among the best-characterized systems for comparing experiments and simulations of protein folding. Recent microsecond-resolution experiments and long duration (totaling milliseconds) single-trajectory modeling have shown that even mechanistic changes in folding kinetics due to mutation can now be analyzed. Thus, a comprehensive set of experimental data would be helpful to benchmark the predictions made by simulations. Here, we use T-jump relaxation in conjunction with protein engineering and report mutational Phi-values (Phi(M)) as indicators for folding transition-state structure of 65 side chain, 7 backbone hydrogen bond, and 6 deletion and /or insertion mutants within loop 1 of the 34-residue hPin1 WW domain. Forty-five cross-validated consensus mutants could be identified that provide structural constraints for transition-state structure within all substructures of the WW domain fold (hydrophobic core, loop 1, loop 2, beta-sheet). We probe the robustness of the two hydrophobic clusters in the folding transition state, discuss how local backbone disorder in the native-state can lead to non-classical Phi(M)-values (Phi(M) > 1) in the rate-determining loop 1 substructure, and conclusively identify mutations and positions along the sequence that perturb the folding mechanism from loop 1-limited toward loop 2-limited folding. (C) 2016 Elsevier Ltd. All rights reserved.

【 授权许可】

Free