A Mutagenic Study of Yeast Actin Conformational Changes

描述（由申请人提供）：肌动蛋白参与许多过程，包括产生收缩力、细胞形状测定、细胞分裂和细胞极性测定。一个正常运作的肌动蛋白细胞骨架，对细胞活力至关重要，取决于一定程度的丝稳定性和肌动蛋白在肌动蛋白丝内的一致性范围，我们的目标是在分子水平上理解影响这些状态的因素。稳定性被认为至少部分受到聚合过程中结合ATP的水解和随后释放Pi以产生ATP肌动蛋白的调节，在迄今为止研究的大多数肌动蛋白中，Pi的释放被延迟，导致纤维稳定性增强，在酵母肌动蛋白中，水解和Pi释放发生在两个过程之间没有明显的间隙。据推测，His73在高级真核生物的肌动蛋白中甲基化，但在酵母中不甲基化，它在延缓Pi的释放中起作用。修饰和未修饰的His之间的pKa差异可能是这两种作用的不同行为的一个因素。使用肌肉和酵母肌动蛋白，我们将研究肌动蛋白聚合和Pi释放作为pH值的函数，以探索H73在控制Pi释放和丝稳定性中的作用。肌动蛋白单体的结晶最常处于“封闭”状态，一旦处于相对于其结构域间隙的“开放”状态，在细丝中，肌动蛋白被观察到处于“封闭”状态，处于“开放”状态，最近处于“倾斜”状态，需要单体的构象扭曲。对于特定肌动蛋白对这些给定状态的相对占有，不同的肌动蛋白结合蛋白如何影响这种状态的分布，以及改变这些状态可能具有的功能意义，我们知之甚少。我们将使用酵母肌动蛋白的定点诱变与酵母表达系统相结合，将突变引入残基中，这些残基被认为是决定肌动蛋白最有可能采用哪一种构象的重要因素，我们将评估蛋白酶易感性、核苷酸交换、我们将使用相同的系统将Cys残基引入特定的位点，然后可以在交联实验中使用，以评估某些预测与某些纤维构象特异性相关的二硫化物形成的可能性。我们将使用类似的方法为顺磁探针创建附着位点，用于EPR实验，以检查肌动蛋白形式相互转化中预测的构象变化。最后，我们将开发使用氢/氘交换方法结合质谱法来评估肌动蛋白对开放和封闭的偏好。

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.