We are using the baculoviral/Sf9 systems to express several unconventional myosins, including myosin III from Limulus, the horseshoe crab, and myosin XV from human. Myosin III is unusual in that there is a kinase domain at the amino-terminus. The myosin is found exclusively in the photoreceptor cells of the visual system. A full-length clone expresses well in Sf9 cells and has been purified to homogeneity. Interestingly, we are unable to detect any MgATPase activity. Recent studies show that it binds only very weakly to actin in either the presence or absence of ATP. The protein is phosphorylated in situ in a circadian manner. We are investigating whether this phosphorylation plays any effect on its activity. Myosin XV is a relatively newly discovered myosin that was identified in mice and humans as a deafness gene. It has a very long proline-rich amino-terminal extension of unknown function which can be alternatively spliced. We are attempting to express three fragments: a fragment that contains the amino-terminus extension through the light chain binding motifs (long S1), a fragment that deletes the alternatively spliced amino-terminus (short S1) and a full-length construct. So far, only the short S1 appears to be expressed in a soluble form. We are currently assessing its enzymatic activity. Myosin VIIb from Drosophila was cloned and we are expressing a fragment containing the head, neck and predicted coiled-coil forming sequences in baculovirus. Preliminary data suggest that the expressed protein is monomeric. It has a Vmax of 6 per sec and a Km of 20 micromolar. We have also expressed a fragment of myosin XVIIIa and are in the process of determining its functional properties. This myosin contains several interesting domains, including an N-terminal KE domain and a PDZ domain. It is alternatively spliced at several places. We are beginning to study the function of the various domains using a combination of biochemical and cell biological techniques
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