Single Molecule Studies of Protein Folding

蛋白质折叠的单分子研究

基本信息

批准号：
8939530
负责人：
William Eaton
金额：
$ 59.24万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

Folding and unfolding rates for the ultrafast folding villin subdomain were determined from a photon-by-photon analysis of fluorescence trajectories in single molecule FRET experiments. One of the obstacles to measuring fast kinetics in single molecule fluorescence experiments is blinking of the fluorophores on a timescale that is not well separated from the process of interest. By incorporating acceptor blinking into a two-state kinetics model, we show that it is possible to extract accurate rate coefficients on the microsecond time scale for folding and unfolding using the maximum likelihood method of I.V. Gopich and A. Szabo. This method yields the most likely parameters of a given model that can reproduce the observed photon trajectories. The extracted parameters agree with both the decay rate of the donor-acceptor cross correlation function and the results of ensemble equilibrium and kinetic experiments using nanosecond laser temperature jump. Kramers developed the theory on how chemical reaction rates are influenced by the viscosity of the medium. At the viscosity of water, the kinetics of unimolecular reactions are described by diffusion of a Brownian particle over a free-energy barrier separating reactants and products. For reactions in solution this famous theory extended Eyring's transition state theory, and is widely applied in physics, chemistry, and biology, including reactions as complex as protein folding. Because the diffusion coefficient of Kramers theory is determined by the dynamics in the sparsely-populated region of the barrier top, its properties have not been directly measured for any molecular system. Here we show that the Kramers diffusion coefficient and free energy barrier can be characterized by measuring the temperature- and viscosity-dependence of the transition path time for protein folding. The transition path is the small fraction of an equilibrium trajectory for a single molecule when the free-energy barrier separating two states is actually crossed. Its duration, the transition path time, can now be determined from photon trajectories for single protein molecules undergoing folding/unfolding transitions5. Our finding of a long transition path time with an unusually small solvent viscosity-dependence suggests that internal friction as well as solvent friction determine the Kramers diffusion coefficient for alpha-helical proteins, as opposed to a breakdown of his theory that occurs for many small-molecule reactions. It is noteworthy that the new and fundamental information concerning Kramers theory and the dynamics of barrier crossings obtained here come from experiments on a protein rather than a much simpler chemical or physical system.

超快折叠式villin子域的折叠率和展开速率是根据单分子FRET实验中荧光轨迹的光子逐光分析确定的。测量单分子荧光实验中快速动力学的障碍之一是闪烁的荧光团在与感兴趣过程没有很好分开的时间表上。通过将受体眨眼融合到两态动力学模型中，我们表明可以使用i.v的最大似然方法提取微秒时间尺度上的准确速率系数，以折叠和展开。 Gopich和A. Szabo。该方法得出可以重现观察到的光子轨迹的给定模型的最可能参数。提取的参数与供体互相关函数的衰减率以及使用纳米秒激光温度跳高的集合平衡和动力学实验的结果一致。克莱默（Kramers）开发了有关化学反应速率如何受培养基粘度影响的理论。在水的粘度下，通过将布朗粒子扩散到分离反应物和产物的自由能屏障上，描述了单分子反应的动力学。对于解决方案中的反应，这种著名的理论扩展了眼环的过渡状态理论，并广泛应用于物理，化学和生物学，包括像蛋白质折叠一样复杂的反应。由于Kramers理论的扩散系数取决于屏障顶部稀疏区域中的动力学，因此尚未直接测量任何分子系统的特性。在这里，我们表明，可以通过测量蛋白质折叠的过渡路径时间的温度和粘度依赖性来表征KRAMERS扩散系数和自由能屏障。当真正越过两个状态的自由能屏障时，过渡路径是单个分子的平衡轨迹的一小部分。现在可以从进行折叠/展开过渡的单蛋白分子的光子轨迹中确定其持续时间，即过渡路径时间。我们发现，具有异常小的溶剂粘度依赖性的长期过渡路径时间表明，内部摩擦以及溶剂摩擦决定了α-螺旋蛋白的Kramers扩散系数，而不是他的理论的分解，而其理论的分解是许多小分子反应发生的。值得注意的是，这里获得的有关Kramers理论的新的和基本的信息来自蛋白质的实验，而不是更简单的化学或物理系统。