A Mutagenic Study of Yeast Actin Conformational Changes

说明(申请人提供)：肌动蛋白参与多种过程，包括收缩力量的产生、细胞形状的确定、胞质分裂和细胞极性的确定。一个功能正常的肌动蛋白细胞骨架对细胞存活至关重要，它依赖于一定程度的微丝稳定性和肌动蛋白在微丝中的顺应性范围，我们的目标是在分子水平上了解影响这些状态的因素。稳定性被认为至少部分受聚合过程中结合的ATP的水解和随后PI的释放以产生ATP肌动蛋白的调节，在迄今研究的大多数肌动蛋白中，PI的释放被延迟，导致细丝稳定性增强。在酵母肌动蛋白中，水解和PI的释放在两个过程之间没有明显的差距。据推测，His73在高等真核生物肌动蛋白中甲基化，而在酵母中不甲基化，在抑制PI释放方面发挥作用。修饰前后组蛋白在PKA上的差异可能是导致这两种肌动蛋白行为不同的原因之一。使用肌肉和酵母肌动蛋白，我们将研究肌动蛋白聚合和PI释放随pH的变化，以探索H73在控制PI释放从而控制细丝稳定性方面的作用。肌动蛋白单体最常见的结晶状态是“闭合”状态，有一次是相对于其结构域间裂隙处于“开放”状态，在细丝中观察到肌动蛋白处于“闭合”状态、“开放”状态，以及最近处于需要单体构象扭曲的“倾斜”状态。关于特定肌动蛋白对这些给定状态的相对占有率，不同的肌动蛋白结合蛋白如何影响这种状态的分布，以及改变这些状态的功能意义可能是什么，人们知之甚少。我们将使用酵母肌动蛋白的定点突变与酵母表达系统相结合，将突变引入残基，这在决定这些构象中哪些肌动蛋白最有可能采用方面是重要的，我们将评估这些变化在蛋白酶易感性、核苷酸交换和聚合方面的影响。我们将使用相同的系统将半胱氨酸残基引入特定的位点，然后用于交联实验，以评估某些预测与某些细丝构象特异相关的二硫化物形成的可能性。我们将使用类似的方法为EPR实验创建顺磁探针的附着位置，以检查在肌动蛋白形式相互转换中预测的构象变化。最后，我们将开发氢/氚交换方法与质谱学相结合的方法来评估肌动蛋白对开放和关闭的偏好。

DESCRIPTION (provided by applicant): Actin is involved in a number of processes including generation of contractile force, cell shape determination, cytokinesis, and determination of cell polarity. A properly functioning actin cytoskeleton, essential for cell viability, depends on a certain degree of filament stability and the range of conform ations that actin can assume within the actin filament, and our goal is to understand at a molecular level, the factors influencing these states. Stability is thought to be at least partially regulated by the hydrolysis of bound ATP during polymerization and the subsequent release of Pi to produce ATP actin, in most actins examined to date, the release of Pi is retarded, leading to enhanced filament stability, in yeast actin, hydrolysis and Pi release occur without a significant gap between the two processes. It has been hypothesized that His73, methylated in higher eukaryotic actins but not in yeast, plays a role in retarding Pi release. The difference in pKa between the modified and unmodified His may be a factor in the different behavior of these two actins. Using both muscle and yeast actin, we will examine actin polymerization and Pi release as a function of pH to explore the role of H73 in controlling Pi release and hence filament stability. The actin monomer has been crystallized most often in a "closed" state and once in an "open" state relative to its interdomain cleft, and in the filament, actin has been observed in a "closed" state, in an "open" state, and more recently in a "tilted" state requiring a conformational twist in the monomer. Little is known about the relative occupancy of these given states by a particular actin, how different actin binding proteins might influence this distribution of states, and what the functional significance of altedng these states might be. We will use site-directed mutagenesis of yeast actin coupled with a yeast expression system to introduce mutations into residues thought to be important in dictating which of these conformations actin is most likely to adopt, and we witl assess the effects of the changes in terms of protease susceptibility, nucieotide exchange, and polymerizabiiityo We will use the same system to introduce Cys residues into specific sites which can then be used in crosslinking experiments to assess the likelihood that certain disulfides, predicted to be specifically associated with certain filament conformations, will form. We will use a similar approach to create attachment sites for paramagnetic probes for EPR experiments to examine conformation changes predicted in the interconversion of actin forms. Finally, we will develop the use of hydrogen/deuterium exchange methods coupled with mass spectrometry to assess the preference of actin for the open vs. closed.