Nome Rene A, Zhao Jason Ming, Hoff Wouter D, Scherer Norbert F
Department of Chemistry, Institute for Biophysical Dynamics, 929 East 57th Street, University of Chicago, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2007 Dec 26;104(52):20799-804. doi: 10.1073/pnas.0701281105. Epub 2007 Dec 19.
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.
我们开展了一项全面的研究，该研究整合了实验和理论非平衡技术，以沿着定义明确的拉伸轴特定坐标绘制能量景观，从而阐明蛋白质展开的机制。沿着光活性黄色蛋白的两个不同轴进行的单分子力-伸长实验，结合非平衡统计力学分析和原子模拟，揭示了能量和机制的各向异性。从实验结果构建的引导分子动力学模拟和自由能曲线表明，沿着一个轴展开呈现出类似过渡态的特征，即六个氢键同时断裂，并在进一步展开过程中观察到弱相互作用。另一个轴呈现出恒定（无峰值）的力分布，表明是非协同过渡，在整个展开过程中焓相互作用（如氢键）被打破。在引导分子动力学计算得出的力-伸长曲线与通过对非平衡功数据进行雅尔津斯基-胡默尔-萨博分析获得的平衡自由能曲线之间发现了惊人的定性一致性。这种各向异性在拉伸距离超过折叠蛋白初始尺寸的两倍以上时仍然存在，这表明到机械上完全展开状态存在丰富的能量景观。我们的发现挑战了协同展开是蛋白质稳定性普遍特征的观点。
