title: Mapping fast protein folding with multiple-site fluorescent probes.
abstract: Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein λ6-85 by engineering into it three fluorescent tryptophan-tyrosine contact probes. The probes report on distances between three different helix pairs: 1-2, 1-3, and 3-2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1-3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same "slow" and "fast" distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1-2 and 3-2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test.
authors.affiliation: Department of Chemistry, University of Illinois, Urbana, IL 61801;
authors.forename: Maxim B
authors.initials: MB
authors.lastname: Prigozhin
authors.affiliation: Department of Physics, University of Illinois, Urbana, IL 61801;
authors.forename: Shu-Han
authors.initials: SH
authors.lastname: Chao
authors.affiliation: Department of Chemistry, University of Illinois, Urbana, IL 61801;
authors.forename: Shahar
authors.initials: S
authors.lastname: Sukenik
authors.affiliation: Department of Chemistry, University of Illinois, Urbana, IL 61801; National Center for Supercomputing Applications, University of Illinois, Urbana, IL 61801; mgruebel@illinois.edu pogorelo@illinois.edu.
authors.forename: Taras V
authors.initials: TV
authors.lastname: Pogorelov
authors.affiliation: Department of Chemistry, University of Illinois, Urbana, IL 61801; Department of Physics, University of Illinois, Urbana, IL 61801; Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801 mgruebel@illinois.edu pogorelo@illinois.edu.
authors.forename: Martin
authors.initials: M
authors.lastname: Gruebele
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D017510:Protein Folding; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D014443:Tyrosine; D014364:Tryptophan
keywords: fluorescence; helix bundle; molecular dynamics; protein folding; thermal denaturation