High-resolution mapping of the folding transition state of a WW domain

Kapil Dave, Marcus Jäger, Houbi Nguyen, Jeffery W. Kelly, Martin Gruebele

Research output: Contribution to journalArticlepeer-review

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 languageEnglish (US)
Pages (from-to)1617-1636
Number of pages20
JournalJournal of Molecular Biology
Volume428
Issue number8
DOIs
StatePublished - 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

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