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

作者：Nome Rene A; Zhao Jason Ming; Hoff Wouter D; Scherer Norbert F^*

来源：Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(52): 20799-20804.

DOI：10.1073/pnas.0701281105

• nonequilibrium dynamics
• photoactive yellow protein
• biophysics

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.

• 出版日期2007-12-26