Protein Folding Kinetics and Activated Rate Models

在这个项目中，PI将研究蛋白质折叠动力学的物理基础：被广泛假设的过渡态理论在多大程度上能够准确地描述蛋白质折叠？为了实现这一目标，将通过定点突变来修饰lambda阻遏蛋白、泛素和磷酸甘油酸激酶，以加快它们在微秒范围内的折叠动力学。初步工作表明，过渡态理论在2微秒以下崩溃。这完全在本研究中使用的温度跳跃仪器的分辨率能力范围内。因此，通常被折叠屏障隐藏的蛋白质集合可以在快速折叠蛋白质中进行检查。初步迹象表明，这种动态是异质的。这项工作将通过探测具有不同光谱特征的多个时间尺度和这样的系综的数量来量化异质性。为了实现在进入折叠状态的过程中解剖蛋白质组的总体目标，将开发一些具体技术：(1)量身定制色氨酸残基附近的电场，并通过定点突变插入电子和质子转移猝灭剂，从而能够对蛋白质光谱性质提供前所未有的控制，从而能够通过快速温度跃升同时探测不同的系谱。(2)多通道红外、吸收和多通道荧光技术的进一步发展将允许大量光谱探测器在微秒和更快的时间尺度上并行应用，从而进一步剖析不同的集合。(3)水在非均相动力学中的作用将通过降低水的密度，并通过监测蛋白质在由此产生的负压下的稳定性和折叠率来探讨。在简单的折叠模型中，水经常被忽略，但目前对折叠时天然蛋白质核心的去溶剂化和“干燥”知之甚少，但这是至关重要的过程。这项研究的主要目的是对新的折叠统计理论进行严格的测试，这些理论的预测在快速时间尺度上与经典模型有很大不同。该理论的基本要素，如无障碍折叠过程中的拉伸系数、玻璃动力学和过渡态前置因素将首次被直接测量。蛋白质折叠的经典生化观点-由屏障分隔的中间状态序列，最终在自然状态-受到基于统计力学的方法的挑战。在这个“新观点”中，蛋白质在到达自然状态的过程中经历了一个粗略的多维能量图景。在折叠过程中，可以探索许多不一定位于一维路径上的蛋白质集合。这项工作将并行应用许多折叠探针，以揭示蛋白质如何在多维能量格局中移动。与理论家的合作将是这项工作的一个核心特征：计算和分析理论现在都能够确定能面的轮廓，但大多数结果都没有经过直接的实验测试。这项工作将培养研究生和本科生。此外，将完成一项将生物物理学原理纳入专业严格的物理化学和物理课程的计划。目前，生物学思想往往主要出现在被淡化的非专业课程中，剥夺了物理和物理化学专业本科生在本科培养中这一重要的应用领域。

In this project, the PI will study the physical foundation of protein folding kinetics: To what extent can the widely assumed transition state theory accurately describe protein folding? To achieve this goal, the proteins lambda repressor, ubiquitin, and phosphoglycerate kinase will be modified by site-directed mutagenesis to speed up their folding kinetics into the microsecond range. Preliminary work has shown that transition state theory breaks down below 2 microseconds. This lies well within the resolution capability of the temperature-jump instrumentation used in this research. As a result, protein ensembles that are normally hidden by a folding barrier can be examined in fast-folding proteins. Preliminary indications are that the dynamics is heterogeneous. This work will quantify the heterogeneity by probing the multiple timescales and the number of such ensembles that have different spectroscopic signatures. A number of specific technologies will be developed to achieve the overall goal of dissecting protein ensembles en route to the folded state: (1) Tailoring the electric field near tryptophan residues, and inserting electron- and proton-transfer quenchers by site-directed mutagenesis, will provide unprecedented control over protein spectral properties, allowing different ensembles to be probed in parallel by fast temperature jumps. (2) Further development of multi-channel infrared, absorption, and multi-channel fluorescence techniques will further dissect different ensembles by allowing large numbers of spectroscopic probes to be applied in parallel on the microsecond and faster time scales. (3) The role of water in the heterogeneous dynamics will be probed by reducing the water density, and by monitoring protein stability and folding rates at the resulting negative pressure. Water is often neglected in simple folding models, but de-solvation and "drying" of the native protein core upon folding are currently little understood, yet are processes of fundamental importance. The main goal of this research is a rigorous test of new statistical theories of folding, whose predictions differ from classical models most dramatically on fast time scales. Fundamental elements of the theory, such as stretching coefficients during barrier-free folding, glassy dynamics, and transition state prefactors will be measured directly for the first time.The classical biochemical view of protein folding - a sequence of intermediate states separated by barriers culminating in the native state - has been challenged by an approach based on statistical mechanics. In this "New View," the protein traverses a rough multidimensional energy landscape on the way to the native state. Many protein ensembles, not necessarily lying on a one-dimensional path, can be explored during folding. This work will apply many folding probes in parallel to reveal how proteins move about on the multidimensional energy landscape. Cooperation with theorists will be a central feature of the work: both computational and analytical theories are now capable of determining the outlines of the energy surface, but most of the results have not been directly tested experimentally. The work will train graduate and undergraduates students. In addition, a program to bring biophysical principles into the rigorous physical chemistry and physics courses taken by majors will be completed. Currently, biological ideas often show up mainly in "watered-down" non-major courses, depriving physics and physical chemistry majors of this important field of application during their undergraduate training.