Myosin VIIa is an unconventional myosin widely expressed in organisms ranging from amoebae to mammals that has been shown to play vital roles in cell adhesion and phagocytosis. We have studied Drosophila myosin VIIa that was expressed in Sf9 cells. We have shown that this myosin has high duty ratio kinetics similar to that of processive motors, but we also showed that it did not easily dimerize even if the full lenght molecule was expressed in Sf9 cells. We have examined the regulation of enzymatic activity of full length myosin VIIa and various C-terminally truncated fragments. Full length myosin VIIa (FLM7a) has a Vmax of about 1 per sec, but has a low apparent affinity for actin, requiring 30-50 uM acin for half-maximal activation. Various C-terminally truncated fragments have a similar Vmax, but much less actin is required for half maximal activation (0.5-1 uM). This means that at 5 uM actin the activity of the fragments is near maximal whereas that of the FLM7a is still barely activated. Removal of even the last 99 amino acids is sufficient to cause this remarkable change in activity. We explored the structural basis for this regulation by using single particle analysis in the electron microscope. We find that in the presence of ATP FLM7a is tightly folded into a compact structure such that the myosin motor domain cannot be discerned whereas in the absence of ATP the molecule is more extended and shows a clearly distinguishable motor domain. Removable of small bits of the tail also is accompanied by the extended conformation leading us to suggest that the compact structure represents an inhibited state of the molecule. We are now searching for binding partners or post-translation modifications that will convert the foled, inactive FLM7a into an extend, active form.[unreadable] [unreadable] Myosin XVIIa has a large predicted coiled-coil forming sequence, but the structure of the myosin has not been examined on a single molecule level. We expressed full length myosin XVIIIa from mouse and showed that it has a long tail similar to that of myosin II. We are examining whether this tail can form filaments.[unreadable] [unreadable] Little is known about the functions of class III unconventional myosins although, with an N-terminal kinase domain, they are potentially both signaling and motor proteins. Limulus myosin III is particularly interesting because it is a phosphoprotein abundant in photoreceptors that becomes more heavily phosphorylated at night by protein kinase A. This enhanced nighttime phosphorylation occurs in response to signals from an endogenous circadian clock and correlates with dramatic changes in photoreceptor structure and function. We seek to understand the role of Limulus myosin III and its phosphorylation in photoreceptors. Here we determined the sites that become phosphorylated in Limulus myosin III and investigated its kinase, actin binding, and myosin ATPase activities. We show that Limulus myosin III exhibits kinase activity and that a major site for both protein kinase A and autophosphorylation is located within loop 2 of the myosin domain, an important actin binding region. We also identify the phosphorylation of an additional protein kinase A and autophosphorylation site near loop 2, and a predicted phosphorylation site within loop 2. We show that the kinase domain of Limulus myosin III shares some pharmacological properties with protein kinase A, and that it is a potential opsin kinase. Finally, we demonstrate that Limulus myosin III binds actin but lacks ATPase activity. We conclude that Limulus myosin III is an actin-binding and signaling protein and speculate that interactions between actin and Limulus myosin III are regulated by both second messenger mediated phosphorylation and autophosphorylation of its myosin domain within and near loop 2.