Abstract
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 Φ-values (ΦM) 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, β-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 ΦM-values (ΦM > 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.
Original language | English (US) |
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Pages (from-to) | 1617-1636 |
Number of pages | 20 |
Journal | Journal of Molecular Biology |
Volume | 428 |
Issue number | 8 |
DOIs | |
State | Published - Apr 24 2016 |
Keywords
- WW domain
- folding transition state
- laser T-jump
- protein folding
- Φ-value analysis
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology