High-Resolution Mapping of the Folding Transition State of a WW Domain. Issue 8 (24th April 2016)

• Record Type: Journal Article
• Authors: Dave, Kapil; Jäger, Marcus; Nguyen, Houbi; Kelly, Jeffery W.; Gruebele, Martin
• Is Part Of: Journal of molecular biology. Volume 428:Issue 8(2016:Apr. 15)
• Page Start: 1617
• Page End: 1636
• Publication Date: 2016-04-24
• Subjects: protein folding -- WW domain -- Φ-value analysis -- folding transition state -- laser T-jump; B factor thermal B factor, a measure for backbone dynamics from X-ray crystal structures -- FBP28 WW WW domain (residues 1–37) derived from mouse formin-binding protein 28 -- FiP hPin1 WW variant in which the wild-type loop 1 sequence (SRSSGR) is replaced by a sequence that folds into a type I G-bulge turn (sequence: SADGR) -- FiP-GTT stabilized FiP variant with the triple-mutation N30G/A31T/S32T that hasten folding of FiP threefold at the thermal midpoint of unfolding -- hPin1 WW WW domain (residues 6–39) derived from human cis/trans-isomerase Pin1 -- hYap65 WW WW domain (residues 1–45) derived from human Yes-associated protein 65 -- MD simulation molecular dynamics simulation -- ΦM mutational phi value, an indicator for structure in the folding transition state -- ΦT temperature-dependent phi value, a parameter for mapping the position of the folding transition state along an entropic reaction coordinate; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2016.02.008
• Languages: English
• ISSNs: 0022-2836

Abstract

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 Φ M ‐values ( Φ 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. Graphical abstract: Highlights: Folding kinetics of a comprehensive set of hPin1 WW mutants, spanning the whole sequence and multiple substitutions at many sites, has been studied. A very conservative phi-value analysis, identifying and excluding disruptive mutations, has revealed the interplay between loops 1 and 2 in the transition state in unprecedented detail. Unusually large "non-classical" phi-values are now explained by local native-state disorder. This comprehensive experimental data set will be valuable for comparison with molecular dynamics simulation, and we begin by creating a hybrid phi-value map for FiP WW domain for comparison with recent all-atom simulations.