Structure, Dynamics and Function of Actin

肌动蛋白是一种主要的细胞蛋白，在肌肉和非肌肉细胞的运动和力量产生中起着关键作用。肌动蛋白多种细胞功能的调控涉及与许多肌动蛋白结合蛋白的特定相互作用。这些相互作用取决于肌动蛋白单体（g -肌动蛋白）和细丝（f -肌动蛋白）在不同构象状态下的平衡。本研究的目的是了解肌动蛋白的动力学和结构状态及其在肌动蛋白基础过程中的作用。该项目的具体目标包括：(i)绘制f -肌动蛋白的分子间界面和这些界面的动态变化；（ii）阐明界面动力学的功能作用；（iii）通过一种主要的细胞肌动蛋白重塑蛋白cofilin确定切断F-actin的结构基础；（iv）测试肌动蛋白单体的开放和封闭核苷酸间隙构象之间ADP/ATP依赖开关的模型。界面动力学的功能作用的研究将包括测量：(i)肌动蛋白结合蛋白的界面扰动，以及（ii）由于界面动力学上的交联约束，肌球蛋白运动产生的变化以及与其他蛋白质相互作用的变化。这些目标将通过多学科和合作研究来实现（使用野生型和突变型蛋白质，用荧光和自旋探针标记半胱氨酸基残基），包括交联、电子显微镜、荧光、质谱、电子顺磁共振、动力学和单分子测量。该项目的更广泛影响将是促进对基本细胞过程的发现和理解，同时为各级学生提供化学/生物界面的跨学科培训-包括来自当地大学代表性不足群体的暑期实习生-并为他们的学术和工业生涯做好准备。

Actin, a major cellular protein, plays a key role in motion and force generation in muscle and non-muscle cells. The regulation of actin's diverse cellular functions involves specific interactions with many actin-binding proteins. These interactions depend on the equilibration of actin monomers (G-actin) and filaments (F-actin) among different conformational states. The objective of this research is to understand the dynamics and structural states of actin and their role in the actin-based processes. The specific aims of this project include: (i) mapping the intermolecular interfaces in F-actin and the dynamic changes at these interfaces; (ii) elucidating the functional role of the interface dynamics; (iii) determining the structural basis of F-actin severing by cofilin, a major cellular actin remodeling protein; and (iv) testing the model of the ADP/ATP dependent switch between the open and closed nucleotide cleft conformations of the actin monomer. Investigations of the functional role of interface dynamics will include measurements of: (i) interface perturbation by actin binding proteins, and (ii) assays of changes in motion generation with myosin and in the interaction with other proteins due to cross-linking constraints on the interface dynamics. These aims will be pursued through multidisciplinary and collaborative studies (using wild type and mutant proteins with cysteinyl residues engineered for labeling with fluorescence and spin probes) involving cross-linking, electron microscopy, fluorescence, mass spectrometry, electron paramagnetic resonance, kinetics, and single molecule measurements.The broader impact of this project will be in advancing the discovery and understanding of basic cellular processes while providing interdisciplinary training in the chemistry/biology interface to students at all levels - including summer trainees from underrepresented groups at the local colleges - and preparing them for academic and industrial careers.