|About the Book|
Actin is a highly conserved and essential protein of all eukaryotic cells that is involved in cell motility, cargo transport, muscle contraction, and cytoskeletal integrity. Actins ability to polymerize, branch, bundle, depolymerize, and interactMoreActin is a highly conserved and essential protein of all eukaryotic cells that is involved in cell motility, cargo transport, muscle contraction, and cytoskeletal integrity. Actins ability to polymerize, branch, bundle, depolymerize, and interact with its over 150 binding partners ( 1), depends on its dynamic properties in the monomer (G-actin) and polymer (F-actin) forms. In this thesis, we first examine the structural transition that actin undergoes when changing from a monomeric (G-actin) to a filamentous (F-actin) state, specifically in the hydrophobic loop, located between subdomains 3 and 4 on actin. We further investigate the hydrophobic loop in the F-actin state, as it has been previously postulated that this loop swings out and inserts into the neighboring actin strand, thus providing stabilizing, lateral interstrand contacts within F-actin. We found using electron paramagnetic resonance (EPR) spin probes and cross-linking methods that the loop resides primarily in a parked conformation in F-actin, docked against its monomer, but swings out to form stabilizing contacts within F-actin. We concluded that the hydrophobic loop is a dynamic element made up of residues that each has an impact upon overall filament dynamics. Assuming that actin stabilizing and destabilizing factors most likely exploit the dynamic fluctuations of actin filaments, we then examined the three elements comprising the hydrophobic pocket of F-actin (C-terminus, hydrophobic loop, DNase I binding loop) in the presence of cofilin, an actin destabilizing protein that severs actin filaments, and phalloidin, an actin stabilizing factor from the fungus A. phalloides. We found that cofilin causes structural shifts in both the hydrophobic loop and the DNase I binding loop and shifts residues in these loops away from the C-terminus within F-actin. In contrast, phalloidin had no significant structural effect on the local environment of spin probes in the two loops. We conclude that the stabilization of F-actin by phalloidin must result from its ability to decrease filament breathing motions and to minimize fluctuations that disrupt F-actin structure. These results provide deeper understanding of the structure and dynamics of actin, and how its dynamics is utilized by other proteins in the cell.