We propose to study the structure and mechanism of action of the UNC-45 molecular chaperone, a myosin- affiliated "UCS domain" protein. Molecular chaperones play key roles in muscle development and function by aiding protein folding and inhibiting protein denaturation and aggregation. We have demonstrated that UNC-45 is critical for skeletal muscle myosin accumulation and myofibril assembly and that it interacts with myosin to protect it from heat-induced aggregation. However, the structure of UNC-45, its mode of interaction with myosin and its roles in skeletal muscle disease are largely unexplored. Our goal is to define the molecular structure of Drosophila UNC-45 and to study its physical interaction with myosin. We will test the hypotheses that 1) ATP binding, which enhances UNC-45 chaperone function, causes structural elements of the protein to undergo conformational change and 2) the UCS domain of UNC-45 interacts with myosin. To this end, we will examine UNC-45 in the apo- and nucleotide-bound states at atomic-level resolution by crystallization, x-ray diffraction and computational analysis. This will be the first high-resolution structure of a UCS domain protein, a class of myosin-associated proteins found in fungi through mammals. We will also image UNC-45 complexed with myosin S-1 by negative staining, electron microscopy and single particle image analysis followed by docking the crystal structures into class averaged projections. This research program will take advantage of our expertise in contractile protein analysis and the capabilities of highly qualified collaborators. Our studies will elucidate the structure of UNC-45, provide an understanding of its interaction with ATP and define ATP- induced conformational changes. Further, our efforts will yield insight into UNC-45's physical interaction with the myosin substrate, which is critical to myofibril assembly and resistance to stress. PUBLIC HEALTH RELEVANCE. The UNC-45 chaperone is critical for the functional integrity of myosin, the molecular motor of muscle. We will study the structure of the UNC-45 chaperone and its interaction with its myosin target. Our efforts will elucidate the mechanism of action of UNC-45 and provide insight into how it functions in normal muscle as well as an understanding of its role during muscle stress.