Protein interactions underlying actin-based motility

描述（由申请人提供）：了解树突状成核和细胞运动性背后的分子相互作用是了解细胞内细胞骨架功能的关键步骤。本项目的目标是鉴定和表征肌动蛋白和肌动蛋白相关蛋白之间的分子相互作用，这些蛋白控制肌动蛋白细胞骨架的形成、组织和动力学。这是一个复杂的系统，因此，重点将主要放在三种特定的蛋白质及其在树突状成核过程中的作用。 具体的目的将是阐明以下的分子细节：（1）profilin与ADF/cofilin结合的单体结合以促进核苷酸交换的动力学，以及profilin结合的肌动蛋白与肌动蛋白丝的倒刺末端的缔合，（2）肌动蛋白丝构象之间的联系，ADF/cofilin的结合动力学，以及ADF/cofilin在丝切断和分解中的作用，（3）阿普2/3复合物与肌动蛋白丝的结合，以及阿普2/3介导的成核和尖端加帽的动力学和热力学。 这些目标将通过结合使用实验生物化学和冷冻电子显微镜技术与完善的计算方法和模型来实现。生物化学工作的目标是详细说明这些蛋白质相互作用的动力学以及系统的其他特性，如肌动蛋白分支的平均长度或肌动蛋白丝的数量。阿普2/3分支丝网络和与ADF/cofilin结合的丝的结构将使用冷冻电子显微镜详细描述。通过使用时间分辨成像，我们将能够捕获结合，分支和切断事件的所有阶段，并使用这些EM重建来产生这些蛋白质如何相互作用的原子级模型。计算工作是这个提议的核心，它的作用是连接这两组实验数据，以了解这些蛋白质的结构与功能之间的关系。原子详细的蛋白质结构和复合物，主要来自我们的电子显微镜重建，将用于布朗动力学模拟，以模拟两个或多个分子的相互作用和缔合动力学。然后将这些结果与生化实验的结果直接进行比较。在并行计算中，我们将计算各种蛋白质之间的结合亲和力，并将这些结果与时间分辨EM工作中观察到的结构变化联系起来。这项工作将提供视角，不仅进入树突状成核，但李斯特菌等细菌的运动，以及整个细胞的功能。

DESCRIPTION (provided by applicant): Understanding the molecular interactions underlying dendritic nucleation and cell motility is a key step in understanding the function of the cytoskeleton within the cell. The goal of this project is to identify and characterize the molecular interactions between actin and actin associated proteins that control the formation, organization and dynamics of the actin cytoskeleton. This is a complicated system, and for that reason, the emphasis will primarily be on three specific proteins and their role in the process of dendritic nucleation. The specific aims will be to elucidate the molecular details of (1) the kinetics of profilin binding to ADF/cofilin-bound monomers to promote nucleotide exchange, and the association of profilin bound actin to the barbed end of actin filaments, (2) the connection between actin filament conformation, the binding kinetics of ADF/cofilin, and the role of ADF/cofilin in filament severing and disassembly, and (3) the binding of Arp 2/3 complex to actin filaments, as well as the kinetics and thermodynamics of Arp 2/3 mediated nucleation, and the capping of pointed ends by the complex. These aims will be achieved through the combined use of experimental biochemistry and cryo-electron microscopy techniques with well-established computational methods and models. The biochemistry work will aim to detail the kinetics of these protein interactions as well as other properties of the system such as the average length of actin branches or numbers of actin filaments. The structure of the Arp 2/3 branched filament networks and filaments bound with ADF/cofilin will be detailed using cryo-electron microscopy. Through the use of time resolved imaging, we will be able to capture all stages of the binding, branching and severing events, and use these EM reconstructions to produce atomic-level models for how these proteins interact. The computational work is central to this proposal, and its role will be to connect these two sets of experimental data to gain an understanding of how the structure relates to the function of these proteins. Atomically-detailed protein structures and complexes, primarily resulting from our electron microscopy reconstructions, will be used in Brownian dynamics simulations to model the interactions and association kinetics of two or more molecules. These results will then be directly compared with the results from the biochemical experiments. In parallel sets of calculations, we will calculate binding affinities between the various proteins are relate these results to the structural changes observed in the time-resolved EM work. This work will give perspective, not only into dendritic nucleation, but the motility of bacteria such as Listeria, and the function of the cell as a whole.