title: Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy.
abstract: In this study we expand the accessible dynamic range of single-molecule force spectroscopy by optical tweezers to the microsecond range by fast sampling. We are able to investigate a single molecule for up to 15 min and with 300-kHz bandwidth as the protein undergoes tens of millions of folding/unfolding transitions. Using equilibrium analysis and autocorrelation analysis of the time traces, the full energetics as well as real-time kinetics of the ultrafast folding of villin headpiece 35 and a stable asparagine 68 alanine/lysine 70 methionine variant can be measured directly. We also performed Brownian dynamics simulations of the response of the bead-DNA system to protein-folding fluctuations. All key features of the force-dependent deflection fluctuations could be reproduced: SD, skewness, and autocorrelation function. Our measurements reveal a difference in folding pathway and cooperativity between wild-type and stable variant of headpiece 35. Autocorrelation force spectroscopy pushes the time resolution of single-molecule force spectroscopy to ∼10 µs thus approaching the timescales accessible for all atom molecular dynamics simulations.
authors.affiliation: Physik Department E22, Technische Universität München, 85748 Garching, Germany.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Žoldák
authors.forename: Johannes
authors.initials: J
authors.lastname: Stigler
authors.forename: Benjamin
authors.initials: B
authors.lastname: Pelz
authors.forename: Hongbin
authors.initials: H
authors.lastname: Li
authors.forename: Matthias
authors.initials: M
authors.lastname: Rief
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics; D008954:Models, Biological; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D052898:Optical Tweezers; D017510:Protein Folding; D013057:Spectrum Analysis / Q000379:methods*; D013816:Thermodynamics
chemical_list: D008840:Microfilament Proteins; C026615:villin
keywords: Chevron plot; Langevin equation; Markov model; optical trapping; thermodynamics