Myosins are actin based molecular motors that have conserved head (motor) and neck (light chain binding) domains and highly divergent tail domains. There is a large family of unconventional myosin genes (i.e. non muscle myosin genes). In collaboration with Dr. Jim Sellers and Dr. Fei Wang at the National Heart Lung and Blood Institute, we are using a baculovirus expression system to purify MYO15 protein consisting of the head and neck domains. The recombinant proteins will be used to study the actin binding, ATP hydrolysis and motility properties of MYO15. To date, two mutations causing deafness have been identified in the myosin-15 head domain (one in mouse and one in humans). The in vitro motor domain assay will provide us with a means of examining the effect of these and future myosin-15 head mutations at the molecular level. The MYO15 tail contains several domains which are candidates for protein interaction motifs. The identification of proteins that functionally interact with MYO15 may provide the best means of determining the role of MYO15 in the auditory system. In addition, interacting proteins are themselves likely to play crucial roles in hearing and would be strong candidates for proteins encoded by DFN loci. We are therefore using the yeast two hybrid system to identify proteins that interact with the MYO15 tail. Positives from the two-hybrid screens will be further examined for biological relevance and promising candidates will be pursued.