Protein Structure and Dynamics from EPR Spectroscopy and MD Simulations

描述（由申请人提供）：该计划项目的长期目标是开发计算工具，可用于从蛋白质的定点自旋标记（SDSL）研究中提取准确的结构和动力学信息。这些新的工具构成了一个必不可少的使能技术，以确定广泛的可溶性和膜结合蛋白质及其复合物的结构和功能动力学。因此，这些工具应该对范德比尔特和其他地方的蛋白质SDSL研究产生直接和重大的影响，我们打算将本项目中开发的所有软件和计算协议免费提供给其他机构的同事。 该项目的具体目标是：1）开发分子动力学和分子模拟协议，使我们能够直接将自旋标记迁移率和可及性的EPR测量与蛋白质结构和结构波动相关联; 2）开发模拟工具和协议，使探针间距离的EPR测量可以直接与蛋白质结构相关联; 3）开发通用计算协议，以便在具体目标1和2中获得的信息可用于建立和改进结构模型;和4）将在目标1-3中开发的工具和策略应用于从T4溶菌酶（已知结构的模型蛋白）获得的SDSL数据，用于CDB 3，一种更复杂的红细胞膜蛋白，迄今为止，其结构仅部分表征，以及淀粉样前体蛋白肽，其结构目前还没有很好地表征。这些特定的蛋白质光谱研究将为开发和测试计算协议提供必要的实验数据。 该计划包括在EPR光谱和蛋白质的SDSL研究（Beth和胡斯特），膜蛋白质结构-功能研究（Beth，Lybrand和Sanders），以及使用分子建模和分子模拟工具从光谱和其他生物物理研究（Lybrand和Smith）获得的距离数据中改进三维蛋白质结构方面具有丰富经验的研究人员。

DESCRIPTION (provided by applicant): The long-term objectives of this program project are to develop computational tools that can be used to extract accurate structural and dynamical information from site-directed spin-labeling (SDSL) studies on proteins. These new tools constitute an essential enabling technology to determine the structures and functional dynamics for a wide range of soluble and membrane-bound proteins and their complexes. As such, these tools should have an immediate and significant impact on SDSL studies of proteins here at Vanderbilt and elsewhere, and we intend to make all software and computational protocols developed in this program project freely available to colleagues at other institutions. The specific aims of this program project are: 1) develop molecular dynamics and molecular modeling protocols to enable us to directly correlate EPR measurements of spin label mobility and accessibility with protein structure and structural fluctuations; 2) develop simulation tools and protocols so that EPR measurements of inter-probe distances can be related directly to protein structure; 3) develop a general computational protocol so that information obtained in Specific Aims 1 and 2 can be used to build and refine structural models; and 4) apply the tools and strategies developed in Aims 1-3 to SDSL data obtained for T4 lysozyme, a model protein of known structure, for CDB3, a more complex erythrocyte membrane protein that is to date only partially characterized structurally, and for amyloid precursor protein peptides that are not well characterized structurally at this time. These specific protein spectroscopic studies will provide the requisite experimental data necessary to develop and test the computational protocols. This program includes investigators with extensive experience in EPR spectroscopy and SDSL studies of proteins (Beth and Hustedt), membrane protein structure-function studies (Beth, Lybrand, and Sanders), and use of molecular modeling and molecular simulation tools to refine three-dimensional protein structures from distance data obtained from spectroscopic and other biophysical studies (Lybrand and Smith).