Date of Degree
PhD (Doctor of Philosophy)
Peter A. Rubenstein
Actin's ability to engage in a wide range of physiological functions requires that it be subject to complex spatial and temporal regulation. This regulation is achieved internally through monomer-monomer contacts and externally through interactions with actin binding proteins. The first part of my thesis focused on better understanding the role of inter-monomeric ionic interactions proposed between subdomains 2 and 3 of opposing monomers in F-actin stabilization. I studied several yeast actin mutants: A167R to disrupt a proposed ionic attraction with R39, A167E to mimic a proposed ionic attraction in muscle actin, and D275R to disrupt a proposed ionic attraction with R39. I investigated the effects of mutations in vivo, effects on filament polymerization characteristics and appearance in vitro, as well as interaction of the mutants with the filament severing protein cofilin. While both in vivo and in vitro data demonstrated the importance of the R39-D275 interaction for yeast actin and the interaction of the filament with cofilin, disruption of this interaction alone did not cause filament fragmentation. Conversely, results with A167 do demonstrate the in vivo and in vitro importance of another potential R39 ionic interaction for filament stabilization.
In the second part of my work I used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry as a tool to gain structural insight into yeast and muscle actin and profilin isoform differences and the actin-profilin interaction. The yeast and muscle actin HD analysis showed greater exchange for yeast G-actin compared to muscle actin in the barbed end pivot region and areas in subdomains 1 and 2, and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared to muscle actin. For yeast-muscle hybrid G-actins, the muscle-like and yeast-like parts of the molecule generally showed exchange characteristics resembling their parent actins. There were a few exceptions to this rule, however: a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3. These exhibited muscle actin-like exchange characteristics even though the areas were yeast-like, suggesting that there is crosstalk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared to both yeast and muscle actins are consistent with mismatched yeast-muscle actin interfaces resulting in decreased stability of the hybrid filament contacts. Actin-profilin HD exchange results demonstrated a possible differential interaction of specific profilin isoforms with specific actin isoforms. While profilin binding mostly caused a decreased exchange for yeast actin peptides, it caused an increase in exchange for muscle actin peptides. Many of the changes observed were in peptides that line or contact the nucleotide cleft, consistent with profilin's ability to alter the kinetics of nucleotide exchange.
actin, actin filament stability, hydrogen deuterium exchange
xiii, 218 pages
Includes bibliographical references (pages 207-218).
Copyright 2009 Ema Stokasimov