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Wan, Qun

Molecular Physiology and Biophysics

2008

PhD

Actin filaments are involved in a wide variety of cellular functions, such as muscle contraction, cell motility, cytokinesis, and endocytosis. ATP hydrolysis plays a key role during its polymerization and depolymerization. To obtain a monomeric form, which is necessary for crystallization, two hydrophobic residues in subdomain 4 (A204E and P243K) were mutated to break hydrophobic interactions in the filament. This nonpolymerizable actin, named AP-actin, was crystallized in both the ADP and ATP states. Nucleotide-dependent conformational changes are only observed in the active site and the sensor loop, but do not propagate to the surface of the four subdomains. The cleft formed by Domain I and II remains closed. Large conformational changes are not observed, perhaps because AP-actin does not have the same actin-actin interactions that occur in the filament. The conformational changes in kinesin obtained by soaking and mutagenesis revealed the important interactions between the P-loop and the Switch II region. The localized changes in the active site of AP-actin between the ATP and ADP states suggest the importance of actin-actin interactions in the filament.