Sequence-dependent folding of single proteins under mechanical load

蛋白质必须折叠成精确的三维结构才能正确发挥功能。折叠过程是细胞中遗传信息从储存到有用部署的重要组成部分。尽管对蛋白质折叠的研究已经进行了几十年，但由于直接测量折叠转变的实验挑战，关于氨基酸序列如何影响折叠仍有很多东西需要了解。本计画将利用光阱所施加的机械力，使单一蛋白质分子重复展开与再折叠，以探讨折叠的基本原理。通过监测平衡（恒定力）和非平衡（变化力）条件下的分子端到端距离，高精度测量单个折叠轨迹。定量的热力学和动力学性质的折叠过程将确定从这些轨迹，包括折叠的自由能，折叠和展开率作为力的函数，存在和位置沿着反应坐标的中间状态，和位置和高度的能量障碍。序列对这些性质的影响将通过改变简单蛋白质结构域的氨基酸序列来系统地研究。

Proteins must fold into precise three-dimensional structures in order to function correctly. The folding process is an essential part of the flow of genetic information in the cell from storage to useful deployment. Despite many decades of research on protein folding, much remains to be learned about how the amino acid sequence affects folding, due to the experimental challenges in measuring the folding transition directly. This project will probe the basic principles of folding by using mechanical force applied by an optical trap to unfold and refold single protein molecules repeatedly. Individual folding trajectories will be measured with high-precision by monitoring the molecular end-to-end distance under both equilibrium (constant force) and non-equilibrium (changing force) conditions. Quantitative thermodynamic and kinetic properties of the folding process will be determined from these trajectories, including the free energy for folding, the folding and unfolding rates as a function of force, the presence and location along the reaction coordinate of intermediate states, and the location and height of energy barriers. The effect of sequence on these properties will be investigated systematically by varying the amino acid sequence of simple protein domains.