title: Axis-dependent anisotropy in protein unfolding from integrated nonequilibrium single-molecule experiments, analysis, and simulation.
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.
authors: Department of Chemistry, Institute for Biophysical Dynamics, 929 East 57th Street, University of Chicago, Chicago, IL 60637, USA.
authors: Rene A
authors: RA
authors: Nome
authors: Jason Ming
authors: JM
authors: Zhao
authors: Wouter D
authors: WD
authors: Hoff
authors: Norbert F
authors: NF
authors: Scherer
mesh_terms: D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry; D001703:Biophysics / Q000379:methods; D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry; D020607:Halorhodospira halophila / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry; D018625:Microscopy, Atomic Force; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D003545:Cysteine