title: High-Resolution Mapping of the Folding Transition State of a WW Domain.
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 (Φ(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, 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.
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Kapil
authors.initials: K
authors.lastname: Dave
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road BCC255, La Jolla, CA 92037, USA; The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road BCC255, La Jolla, CA 92037, USA.
authors.forename: Marcus
authors.initials: M
authors.lastname: Jäger
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Houbi
authors.initials: H
authors.lastname: Nguyen
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road BCC255, La Jolla, CA 92037, USA; The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road BCC255, La Jolla, CA 92037, USA.
authors.forename: Jeffery W
authors.initials: JW
authors.lastname: Kelly
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. Electronic address: mgruebel@illinois.edu.
authors.forename: Martin
authors.initials: M
authors.lastname: Gruebele
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000595:Amino Acid Sequence; D017353:Gene Deletion; D006801:Humans; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature; D013816:Thermodynamics; D014157:Transcription Factors; D000091102:YAP-Signaling Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D010750:Phosphoproteins; D014157:Transcription Factors; D000091102:YAP-Signaling Proteins; C088374:YAP1 protein, human; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase
keywords: WW domain; folding transition state; laser T-jump; protein folding; Φ-value analysis