Axis-dependent anisotropy in protein unfolding from integrated nonequilibrium single-molecule experiments, analysis, and simulation

doi:10.1073/pnas.0701281105

Axis-dependent anisotropy in protein unfolding from integrated nonequilibrium single-molecule experiments, analysis, and simulation

*Department of Chemistry,
^†Institute for Biophysical Dynamics, 929 East 57th Street, University of Chicago, Chicago, IL 60637;
^‡Department of Radiology, Johns Hopkins University, Baltimore, MD 21205; and
^§Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078

Edited by William A. Eaton, National Institutes of Health, Bethesda, MD, and approved November 7, 2007 (received for review February 12, 2007)

Abstract

We present a comprehensive study that integrates experimental and theoretical nonequilibrium techniques to map energy landscapes along well defined pull-axis specific coordinates to elucidate mechanisms of protein unfolding. Single-molecule force-extension experiments along two different axes of photoactive yellow protein combined with nonequilibrium statistical mechanical analysis and atomistic simulation reveal energetic and mechanistic anisotropy. Steered molecular dynamics simulations and free-energy curves constructed from the experimental results reveal that unfolding along one axis exhibits a transition-state-like feature where six hydrogen bonds break simultaneously with weak interactions observed during further unfolding. The other axis exhibits a constant (unpeaked) force profile indicative of a noncooperative transition, with enthalpic (e.g., H-bond) interactions being broken throughout the unfolding process. Striking qualitative agreement was found between the force-extension curves derived from steered molecular dynamics calculations and the equilibrium free-energy curves obtained by Jarzynski–Hummer–Szabo analysis of the nonequilibrium work data. The anisotropy persists beyond pulling distances of more than twice the initial dimensions of the folded protein, indicating a rich energy landscape to the mechanically fully unfolded state. Our findings challenge the notion that cooperative unfolding is a universal feature in protein stability.

Footnotes

^¶To whom correspondence should be addressed. E-mail: nfschere{at}uchicago.edu
Author contributions: R.A.N., J.M.Z., W.D.H., and N.F.S. designed research; R.A.N. and J.M.Z. performed research; R.A.N., J.M.Z., and N.F.S. analyzed data; and R.A.N., J.M.Z., W.D.H., and N.F.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0701281105/DC1.