A Mutagenic Study of Yeast Actin Conformational Changes

性状（由申请人提供）：肌动蛋白参与收缩力的产生、细胞形状的确定、胞质分裂和细胞极性的确定。肌动蛋白的正常功能是细胞生存的关键，它依赖于肌动蛋白丝的稳定性和构象的灵活性。肌动蛋白单体的动态性质，无论是游离的还是在肌动蛋白丝内，都是肌动蛋白变构调节所必需的，无论是内部的还是由一系列肌动蛋白调节蛋白施加的。这种调节的一个表现是酵母与肌肉肌动蛋白的功能行为的差异。它们有87%的相同性，并且具有几乎可重叠的结构。然而，它们的参数不同，如聚合的速度，倾向的丝片段，核苷酸交换率，磷酸盐释放率后结合核苷酸水解（丝动力学的决定因素），激活肌球蛋白的能力，并响应肌动蛋白结合蛋白，如cofilin和Arp 2/3复合物。为了在分子水平上理解肌动蛋白丝动力学的调节方式，必须首先理解蛋白质内构象变化的传递方式，以及这些通信网络的差异如何导致肌动蛋白亚型的行为差异。我们将使用一系列的酵母/肌肉杂交肌动蛋白，通过定点诱变制造，以更好地了解肌动蛋白的两个结构域在控制肌动蛋白单体的行为在G-形式和长丝中的作用。与酵母作为一个模型系统，我们将扩展我们的初步工作与这样的混合蛋白质使用细胞生物学和蛋白质化学方法的组合，如聚合动力学，共价交联，和氢/氘交换，以解决以下目标。我们将描绘一个子域1列的氨基酸在调节域/域相互作用的作用。我们将评估丝断裂对细胞骨架功能的影响，以及内在丝稳定性和cofilin切断之间的平衡如何影响细胞骨架行为。我们将研究两个提议的单体间离子相互作用对细丝稳定性的重要性，我们将评估两个关键的亚结构域4残基对亚结构域1和2在与肌动蛋白结合蛋白相互作用中的行为的作用。健康意义：这项工作将为肌动蛋白调节的分子基础提供新的见解，当异常时，会导致癌症，肌肉疾病和听力损失等病理状态。

DESCRIPTION (provided by applicant): Actin is involved in generation of contractile force, cell shape determination, cytokinesis, and determination of cell polarity. Proper actin fuction, essential for cell viability, depends on a certain degree of filament stability and conformational flexibility. The dynamic nature of the actin monomer, both free and within the actin filament, is required for allosteric regulation of actin, both internally, and imposed upon it by an array of actin regulatory proteins. A manifestation of this regulation is the difference in functional behavior of yeast vs. muscle actin. They are 87% identical and have virtually superimposable structures. Yet they differ in parameters such as speed of polymerization, propensity of filaments to fragment, rate of nucleotide exchange, rate of phosphate release following bound nucleotide hydrolysis (a determinant of filament dynamics), ability to activate myosin, and response to actin binding proteins such as cofilin and Arp2/3 complex. To understand at the molecular level, the ways in which actin filament dynamics are regulated, one must first understand the way in which conformational changes are transmitted within the protein and how differences in these communication networks result in the behavioral differences of actin isoforms. We will use a series of yeast/muscle hybrid actins, manufactured by site-directed mutagenesis, to better understand the roles of actin's two domains in controlling the behavior of the actin monomer in the G-form and in the filament. With yeast as a model system, we will extend our initial work with such hybrid proteins using a combination of cell biological and protein chemical approaches such as polymerization kinetics, covalent crosslinking, and hydrogen/deuterium exchange to address the following aims. We will delineate the role of a subdomain 1 column of amino acids in regulation of domain/domain interactions. We will assess the effect of filament fragmentation on cytoskeletal function and how the balance between inherent filament stability and cofilin severing affects cytoskeletal behavior. We will examine the importance of two proposed intermonomer ionic interactions on filament stability, and we will assess the role of two critical subdomain 4 residues on the behavior of subdomains 1 and 2 in their interaction with actin binding proteins. Health Significance: This work will provide new insight into the molecular basis of actin regulation which, when abnormal, leads to pathological states such as cancer, muscle disease, and hearing loss.