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.
| Lee, Cho-Yin; Lou, Jizhong; Wen, Kuo-Kuang et al. (2016) Regulation of actin catch-slip bonds with a RhoA-formin module. Sci Rep 6:35058 |
| Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema et al. (2011) Mutant profilin suppresses mutant actin-dependent mitochondrial phenotype in Saccharomyces cerevisiae. J Biol Chem 286:41745-57 |
| Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S et al. (2011) Functional adaptation between yeast actin and its cognate myosin motors. J Biol Chem 286:30384-92 |
| Kudryashov, Dmitri S; Grintsevich, Elena E; Rubenstein, Peter A et al. (2010) A nucleotide state-sensing region on actin. J Biol Chem 285:25591-601 |
| Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema et al. (2010) A potential yeast actin allosteric conduit dependent on hydrophobic core residues val-76 and trp-79. J Biol Chem 285:21185-94 |
| Scoville, Damon; Stamm, John D; Altenbach, Christian et al. (2009) Effects of binding factors on structural elements in F-actin. Biochemistry 48:370-8 |
| Stokasimov, Ema; Rubenstein, Peter A (2009) Actin isoform-specific conformational differences observed with hydrogen/deuterium exchange and mass spectrometry. J Biol Chem 284:25421-30 |
| Wen, Kuo-Kuang; Rubenstein, Peter A; DeMali, Kris A (2009) Vinculin nucleates actin polymerization and modifies actin filament structure. J Biol Chem 284:30463-73 |
| Stokasimov, Ema; McKane, Melissa; Rubenstein, Peter A (2008) Role of intermonomer ionic bridges in the stabilization of the actin filament. J Biol Chem 283:34844-54 |
| Wen, Kuo-Kuang; McKane, Melissa; Houtman, Jon C D et al. (2008) Control of the ability of profilin to bind and facilitate nucleotide exchange from G-actin. J Biol Chem 283:9444-53 |
Showing the most recent 10 out of 58 publications
