Molecular Simulations of Cotranslational Folding

描述（由申请人提供）：当前分子生物学的一个重要目标是了解蛋白质的合成和折叠如何相互耦合。尽管许多大分子组分的高分辨率结构有助于理解核糖体合成蛋白质时发生的事件，但目前只有低分辨率的核糖体新生链复合物（rna）的结构和动力学视图。更重要的是，基本上所有的RNC研究都是在RNC停止的条件下进行的，即在其构象动力学有效地处于热力学平衡的条件下进行的。然而，越来越多的证据表明，新生蛋白质的命运在很大程度上取决于翻译发生的速度，这意味着新合成的蛋白质在错误折叠和天然构象之间的分配至少部分受到动力学控制。因此，迫切需要能够在主动翻译过程中对rnc进行结构表征的方法。由于实验表征可能仍然是一个棘手的问题，因此建议在这里使用分子模拟方法代替。因此，概述了开发一个模拟框架的工作计划，该框架可以准确地模拟细菌核糖体中的耦合合成折叠事件，并可以完全定义其附着的伴侣，触发因子（TF）在分离的单体和完整的多体模型中的作用。将追求三个具体目标。首先，将使用显式溶剂分子动力学模拟来确定触发因子单独和与核糖体复合的构象灵活性的程度。这些研究将确定TF在其功能相关状态下的构象适应性的限制，并将为开发TF- rnc的现实模拟模型提供基础。其次，将开发一个精确的粗粒度（CG）模拟模型，使停滞的TF-RNC配合物的构象行为可以直接建模；适当地参数化，该模型将使tf - rnc的广泛实验观察在真正的结构水平上合理化。最后，CG模拟模型将用于模拟单体和多体中主动翻译RNC复合物（带或不带TF）的共翻译折叠事件。后一项研究将提供新生蛋白链的结构和动态图像，从它们从核糖体出口通道出现的那一刻起，到它们完成折叠或错误折叠的那一刻，这是传统实验方法无法实现的。因此，拟议的研究有望大大提高对影响新生蛋白质在其翻译过程中折叠或错误折叠倾向的因素的理解。

DESCRIPTION (provided by applicant): An important current goal in molecular biology is to understand how the synthesis and folding of proteins are coupled to each other. Although the understanding of events that occur as a protein is synthesized by the ribosome has been aided by high-resolution structures of many of the macromolecular components, only low-resolution views of the structure and dynamics of ribosome nascent-chain complexes (RNCs) are currently available. More importantly, essentially all studies of RNCs have been performed under conditions in which the RNC is stalled, i.e. under conditions in which its conformational dynamics are effectively at thermodynamic equilibrium. There is increasing evidence, however, that the fates of nascent proteins can depend significantly on the rate at which translation occurs, which implies that the partitioning of a newly synthesized protein between misfolded and native conformations is at least partly under kinetic control. There is, therefore, an urgent need for methods that can structurally characterize RNCs during active translation. Since experimental characterization is likely to remain an intractable problem it is proposed here to use molecular simulation methods instead. A plan of work is therefore outlined for developing a simulation framework that can accurately model coupled synthesis-folding events in the bacterial ribosome and that can fully define the role of its attached chaperone, trigger factor (TF), both in isolated monosomes and in models of complete polysomes. Three Specific Aims will be pursued. First, explicit-solvent molecular dynamics simulations will be used to determine the extent of trigger factor's conformational flexibility alone and in complex with the ribosome. These studies will establish the limits of TF's conformational adaptability in its functionally relevant states and will provide the basis for developing a realistic simulation model of TF-RNCs. Second, an accurate coarse-grained (CG) simulation model will be developed that allows the conformational behavior of stalled TF-RNC complexes to be directly modeled; properly parameterized, this model will enable a wide range of experimental observations on TF-RNCs to be rationalized at a truly structural level. Finally, the CG simulation model will be used to simulate cotranslational folding events in actively translating RNC complexes (with and without TF) in monosomes and polysomes. These latter studies will provide structural and dynamic pictures of nascent protein chains from the moment that they emerge from the ribosome's exit tunnel to the moment that they complete folding or misfolding in a way that is not achievable by conventional experimental methods. As such, the proposed studies are expected to greatly improve understanding of the factors that affect a nascent protein's propensity to fold or misfold during the course of its translation.