Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 428, Issue 8, Pages 1617-1636Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2016.02.008
Keywords
protein folding; WW domain; Phi-value analysis; folding transition state; laser T-jump
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Funding
- German Research Council (DFG)
- La Jolla Interfaces in Science (LJIS) training program
- [R01 GM 93318]
- [GM051105]
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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.
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