The best-studied molecular motors are the muscle myosins, which work in filamentous assemblies. Here we focus on unconventional myosin V, which functions as a monomer or in small numbers to transport vesicles. A comparative approach allows us to identify common principles among molecular motors, as well as to understand how variations in the kinetic cycle adapt a particular class of motors for its cellular role. The lessons learned from the regulation and function of myosin II will be applied to the design and interpretation of the experiments proposed here. The goal of specific aim #1 is to use enzymatic and in vitro motility assays to understand the molecular mechanism by which myosin V's motor activity is regulated by calcium binding to calmodulin, which binds to 6 JO motifs (consensus sequence lQxxxRGxxxR) in the neck region of myosin V. Cryoelectron microscopy will be used to assess the structural impact of calcium binding. In specific aim #2, we will investigate the mechanism of myosin V processivity. Transient kinetics of wild type and mutant expressed myosin Vs will be used to understand the kinetic and structural features whereby the two heads of myosin V can be coordinated. Optical trapping studies (collaboration with Dr. Warshaw) will complement the biochemical studies. We will also visualize myosin V bound to actin in the presence of ATP by cryo-electron microscopy (collaboration with Drs. Hanein and Volkmann), and identify the structural elements that account for this tight binding. Specific aim #3 is to express and isolate a small oligomer of actin for crystallization studies. We have recently been successful in developing a high-yield expression system for muscle actin. We will express mutants designed to terminate polymerization at the dimeric (or tetrameric) state for studies of the native actin-actin interface, and for potential co-crystallization efforts with myosin subfragments (collaboration with Dr. Rould). The overall goal is to exploit the unique features of other members of the myosin superfamily to more fully understand the principles by which molecular motors are designed