A Mutagenic Study of Yeast Actin Conformational Changes

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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.