Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
J Biol Chem. 2011 Sep 9;286(36):31072-9. doi: 10.1074/jbc.M111.264093. Epub 2011 Jul 16.
Understanding protein dynamics requires a comprehensive knowledge of the underlying potential energy surface that governs the motion of each individual protein molecule. Single molecule mechanical studies have provided the unprecedented opportunity to study the individual unfolding pathways along a well defined coordinate, the end-to-end length of the protein. In these experiments, unfolding requires surmounting an energy barrier that separates the native from the extended state. The calculation of the absolute value of the barrier height has traditionally relied on the assumption of an attempt frequency, υ(‡). Here we used single molecule force-clamp spectroscopy to directly determine the value of υ(‡) for mechanical unfolding by measuring the unfolding rate of the small protein ubiquitin at varying temperatures. Our experiments demonstrate a significant effect of the temperature on the mechanical rate of unfolding. By extrapolating the unfolding rate in the absence of force for different temperatures, varying within the range spanning from 5 to 45 °C, we measured a value for the activation barrier of ΔG(‡) = 71 ± 5 kJ/mol and an exponential prefactor υ(‡) ∼4 × 10(9) s(-1). Although the measured prefactor value is 3 orders of magnitude smaller than the value predicted by the transition state theory (∼6 × 10(12) s(-1)), it is 400-fold higher than that encountered in analogous experiments studying the effect of temperature on the reactivity of a protein-embedded disulfide bond (∼10(7) M(-1) s(-1)). This approach will allow quantitative characterization of the complete energy landscape of a folding polypeptide from highly extended states, of capital importance for proteins with elastic function.
了解蛋白质动力学需要全面了解控制每个蛋白质分子运动的潜在能量表面。单分子力学研究为研究沿着明确定义的坐标（蛋白质的首尾长度）的单个展开途径提供了前所未有的机会。在这些实验中，展开需要克服将天然状态与伸展状态分开的能量障碍。障碍高度的绝对值的计算传统上依赖于尝试频率 υ(‡)的假设。在这里，我们使用单分子力钳光谱法通过测量在不同温度下的小蛋白泛素的展开速率来直接确定机械展开过程中 υ(‡)的价值。我们的实验表明温度对机械展开速率有显著影响。通过在没有力的情况下外推不同温度下的展开速率，温度范围从 5 到 45°C 不等，我们测量了激活障碍 ΔG(‡)的值为 71 ± 5 kJ/mol 和指数前因子 υ(‡)∼4 × 10(9) s(-1)。尽管测量的前因子值比过渡态理论预测的值（∼6 × 10(12) s(-1)）小 3 个数量级，但它比在类似的研究蛋白质嵌入二硫键的温度对反应性影响的实验中遇到的值（∼10(7) M(-1) s(-1)）高 400 倍。这种方法将允许从高度伸展状态对折叠多肽的完整能量景观进行定量描述，对于具有弹性功能的蛋白质至关重要。
