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的产生中起着关键作用。肌动蛋白的多种细胞功能的调节涉及到与许多肌动蛋白结合蛋白的特定相互作用。这些相互作用依赖于肌动蛋白单体(G-肌动蛋白)和细丝(F-肌动蛋白)在不同构象状态之间的平衡。本研究的目的是了解肌动蛋白的动力学和结构状态，以及它们在基于肌动蛋白的过程中的作用。该项目的具体目标包括：(I)绘制F-肌动蛋白的分子间界面以及这些界面上的动态变化；(Ii)阐明界面动力学的功能作用；(Iii)确定F-肌动蛋白被主要的细胞肌动蛋白重塑蛋白cofilin切断的结构基础；以及(Iv)测试肌动蛋白单体的开放和闭合核苷酸裂解构象之间的ADP/ATP依赖转换模型。对界面动力学的功能作用的研究将包括测量：(I)肌动蛋白结合蛋白对界面的扰动，以及(Ii)分析肌球蛋白运动产生的变化以及由于界面动力学上的交联限制而与其他蛋白质相互作用的变化。这些目标将通过多学科和协作研究(使用带有半胱氨基残基的野生型和突变蛋白质，利用荧光和自旋探针标记)，涉及交联体、电子显微镜、荧光、质谱学、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--包括来自当地大学中代表性不足的群体的暑期实习生--提供化学/生物界面的跨学科培训，并为他们的学术和工业职业生涯做好准备。

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.