Axis-dependent anisotropy in protein unfolding from integrated nonequilibrium single-molecule experiments, analysis, and simulation
Axis-dependent anisotropy in protein unfolding from integrated nonequilibrium single-molecule experiments, analysis, and simulation
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DOI：
10.1073/pnas.0701281105
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发表时间：
2007-12-26
影响因子：
11.1
通讯作者：
Scherer, Norbert F.
中科院分区：
文献类型：
--
作者：
Nome, Rene A.;Zhao, Jason Ming;Scherer, Norbert F.
我们提出了一项综合研究，整合了实验和理论非平衡技术，沿着明确定义的拉轴特定坐标绘制能量图，以阐明蛋白质展开的机制。沿光敏黄色蛋白两个不同轴的单分子力延伸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械各向异性。根据实验结果构建的引导分子动力学模拟和自由能曲线表明，沿一个轴的展开呈现出一种类似过渡态的特征，其中六个氢键同时断裂，并在进一步展开过程中观察到弱相互作用。另一个轴表现出恒定（未达到峰值）的力分布，表明非合作转变，在整个展开过程中，焓（例如，氢键）相互作用被破坏。在由导向分子动力学计算得出的力-延伸曲线与通过非平衡工作数据的 Jarzynski-Hummer-Szabo 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。各向异性持续超过折叠蛋白质初始尺寸两倍以上的拉动距离，表明机械完全展开状态具有丰富的能量景观。我们的研究结果挑战了“合作解折叠是蛋白质稳定性的普遍特征”这一观点。
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.