Molecular Simulations of Cotranslational Folding

描述（由申请人提供）：分子生物学目前的一个重要目标是了解蛋白质的合成和折叠是如何相互耦合的。尽管对蛋白质由核糖体合成时发生的事件的理解得到了许多大分子组分的高分辨率结构的帮助，但目前只有核糖体新生链复合物（RNCs）的结构和动力学的低分辨率视图。更重要的是，基本上所有RNC的研究都是在RNC停滞的条件下进行的，即在其构象动力学有效地处于热力学平衡的条件下。然而，越来越多的证据表明，新生蛋白质的命运可以显着依赖于翻译发生的速率，这意味着一个新合成的蛋白质之间的错误折叠和天然构象的分区至少部分是在动力学控制。因此，有一个迫切需要的方法，可以在结构上表征RNCs在主动翻译。由于实验表征可能仍然是一个棘手的问题，因此这里建议使用分子模拟方法来代替。因此，概述了一个计划的工作，开发一个模拟框架，可以准确地模拟耦合的合成折叠事件的细菌核糖体，并可以充分定义其所附的伴侣，触发因子（TF）的作用，无论是在孤立的单体和完整的多聚核糖体模型。将追求三个具体目标。首先，显式溶剂分子动力学模拟将被用来确定触发因子的构象灵活性的程度，单独和复杂的核糖体。这些研究将建立TF的构象适应性在其功能相关的状态的限制，并将提供一个现实的TF-RNC模拟模型的基础。其次，将开发一个精确的粗粒度（CG）模拟模型，允许停滞的TF-RNC复合物的构象行为直接建模;适当参数化，该模型将使广泛的TF-RNC的实验观察在一个真正的结构水平合理化。最后，CG模拟模型将用于模拟单体和多核糖体中主动翻译RNC复合物（有和没有TF）的共翻译折叠事件。后者的研究将提供新生蛋白质链的结构和动态图片，从它们从核糖体的出口隧道出现的那一刻起，到它们以常规实验方法无法实现的方式完成折叠或错误折叠的那一刻。因此，预计拟议的研究将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.