Molecular simulation of protein folding in vivo

描述（由申请人提供）：分子生物物理学的一个重要的当前目标是确定在体外观察到的蛋白质行为在多大程度上反映了所发生的行为 in vivo.基于“细胞内”NMR和荧光技术的实验方法正开始弥合知识差距，但此类研究在执行、分辨率和解释方面可能存在重大技术挑战;此外，它们通常一次只允许研究一种或几种类型的大分子。为了提供这些实验方法的补充，本建议的目的是开发一种布朗动力学（BD）模拟方法，能够在近原子水平的分辨率建模细胞内环境，并应用该方法模拟蛋白质行为的关键方面在原核细胞大肠杆菌的细胞质模型。模拟方法将允许所有的大分子被视为完全灵活的，从而允许蛋白质折叠事件在体内进行建模，并将提供一个严格的建模的流体动力学相互作用，这是至关重要的，包括如果要准确地捕捉大分子的扩散特性。将实现三个具体目标。在目标1，一个全面的，并行化的粗粒度（CG）BD模拟方法将完成，允许大规模的生物分子系统进行建模。拟议的工作将涉及（a）一种新的方法，用于在一个非常大的规模上模拟流体动力学相互作用的实施，和（B）并行化的模拟代码，使其有效地运行在共同的分布式内存计算机集群。在目标2中，将导出用于蛋白质/RNA系统的CG模拟的综合力场，以用于目标1中开发的模拟代码。重要的是，力场的参数化将以两种根本不同的方式进行：“自上而下”，使用弱大分子相互作用热力学的实验数据，和“自下而上”，使用全原子，显式溶剂分子动力学模拟数据。除了被参数化的全面的方式，派生的CG力场将是唯一的，也有其流体动力学参数明确参数化。最后，在目标3中，目标1和2中开发的方法将用于进行一系列模拟研究，研究在体内遇到的高度拥挤条件下蛋白质行为的基本方面。BD模拟的蛋白质折叠热力学在浓缩的单蛋白质溶液中进行比较的H/D交换测量的结果。BD模拟蛋白质在大肠杆菌胞质中的扩散。大肠杆菌的目标是在计算机上再现“细胞内”NMR和荧光恢复后光漂白（FRAP）实验的结果，这些实验也在大肠杆菌中进行。杆菌最后，用BD模拟方法对蛋白质在大肠杆菌中折叠的热力学和动力学进行了模拟。coli细胞质中的蛋白质，并与相应的实验数据进行比较。如果成功的话，这里开发和应用的方法将提供一个从根本上新的大分子在体内的行为，将能够合理化以前不太了解的实验结果，并作出直接可测试的预测。模拟代码及其伴随的力场将免费提供给社区。

DESCRIPTION (provided by applicant): An important current goal in molecular biophysics is to determine the extent to which protein behavior observed in vitro is reflective of that occurring in vivo. Experimental approaches based on "in-cell" NMR and fluorescence techniques are beginning to bridge the gaps in knowledge, but such studies can present significant technical challenges in terms of execution, resolution and interpretation; in addition, they typically allow only one or a few types of macromolecule to be studied at a time. To provide a complement to these experimental approaches, the purpose of this proposal is to develop a Brownian dynamics (BD) simulation method capable of modeling intracellular environments at a near-atomic level of resolution, and to apply the method to simulate key aspects of protein behavior in a model of the cytoplasm of the prokaryote Escherichia coli. The simulation method will allow all macromolecules to be treated as fully flexible, thereby allowing protein folding events in vivo to be modeled, and will provide a rigorous modeling of the hydrodynamic interactions that are crucial to include if the diffusional properties of macromolecules are to be accurately captured. Three Specific Aims will be pursued. In Aim 1, a comprehensive, parallelized coarse-grained (CG) BD simulation method will be completed that allows large-scale biomolecular systems to be modeled. The proposed work will involve (a) the implementation of a novel method for modeling hydrodynamic interactions on a very large scale, and (b) parallelization of the simulation code so that it runs efficiently on common distributed-memory computer clusters. In Aim 2, a comprehensive force field for use in CG simulations of protein/RNA systems will be derived for use with the simulation code developed in Aim 1. Importantly, parameterization of the force field will be performed in two fundamentally different ways: 'top down', using experimental data on the thermodynamics of weak macromolecular interactions, and 'bottom up', using all-atom, explicit-solvent molecular dynamics simulation data. In addition to being parameterized in a comprehensive way, the derived CG force field will be unique in also having its hydrodynamic parameters explicitly parameterized. Finally, in Aim 3, the methods developed in Aims 1 and 2 will be used to perform a series of simulation studies examining fundamental aspects of protein behavior in the highly crowded conditions encountered in vivo. BD simulations of protein folding thermodynamics in concentrated single-protein solutions will be compared with the results of H/D exchange measurements. BD simulations of protein diffusion in a model of the cytoplasm of E. coli will be aimed at reproducing in silico the results of 'in-cel' NMR and fluorescence-recovery-after-photobleaching (FRAP) experiments, also performed in E. coli. Finally, BD simulations of the thermodynamics and kinetics of protein folding in the E. coli cytoplasm will also be carried out and compared with corresponding experimental data. If successful, the methods developed and applied here will provide a fundamentally new view of macromolecular behavior in vivo that will be capable of rationalizing previously poorly understood experimental results and making directly testable predictions. Both the simulation code and its attendant force fields will be made freely available to the community.