A Mutagenic Study of Yeast Actin Conformational Changes

描述(申请人提供)：肌动蛋白参与收缩力量的产生、细胞形状的确定、胞质分裂和细胞极性的确定。适当的肌动蛋白功能是细胞存活所必需的，依赖于一定程度的微丝稳定性和构象灵活性。肌动蛋白单体的动态性质，无论是游离的还是在肌动蛋白细丝内的，都是肌动蛋白变构调节所必需的，无论是在内部，还是由一系列肌动蛋白调节蛋白强加于肌动蛋白。这种调节的一个表现是酵母和肌肉肌动蛋白在功能行为上的差异。它们87%相同，具有几乎可重叠的结构。然而，它们在聚合速度、细丝断裂倾向、核苷酸交换速率、结合核苷酸水解后的磷酸盐释放速率(细丝动力学的决定因素)、激活肌球蛋白的能力以及对肌动蛋白结合蛋白(如Cofilin和Arp2/3复合体)的反应等参数上存在差异。要在分子水平上了解肌动蛋白细丝动力学的调控方式，首先必须了解构象变化在蛋白质内传递的方式，以及这些通讯网络中的差异如何导致肌动蛋白亚型的行为差异。我们将使用一系列通过定点突变制造的酵母/肌肉杂交肌动蛋白，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在G型和细丝中的行为中的作用。以酵母为模型系统，我们将使用细胞生物学和蛋白质化学的组合方法，如聚合动力学、共价交联和氢/氘交换，来扩展我们对此类杂交蛋白的初步工作，以实现以下目标。我们将描述亚域1氨基酸在结构域/结构域相互作用调节中的作用。我们将评估细丝断裂对细胞骨架功能的影响，以及内在细丝稳定性和粘连蛋白切断之间的平衡如何影响细胞骨架的行为。我们将研究两个拟议的单体间离子相互作用对细丝稳定性的重要性，并评估两个关键的亚区4残基在亚区1和亚区2与肌动蛋白结合蛋白相互作用中的作用。健康意义：这项工作将为肌动蛋白调控的分子基础提供新的见解，当肌动蛋白调控异常时，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.