The overall aim of the project is to determine atomic structures of proteins critical for muscle contraction and its regulation, including regions that can be selectively targeted by small molecule drugs. X-ray crystallography, supplemented by biochemical techniques and computer analyses, will be the primary methods used. Atomic resolution structures of the myosin head have revealed various states of the contractile cycle. A major aim is to obtain high-resolution structural information for the off-state of molluscan, vertebrate smooth muscle, and other related regulated myosins, with a focus on a nearly complete myosin and on heavy meromyosin(HMM), a large soluble regulated subfragment. Since regions of vertebrate smooth muscle myosin containing the regulatory light chain (RLC) have not yet been crystallized, specialized regulatory domains containing truncated yet phosphorylatable RLC's will also be studied, in order to compare the conformations of this domain in myosins regulated by different triggers. Our understanding of thin filament structure and its stabilization will be improved by the second aim of the project, to extend crystallographic analyses of tropomyosin to smooth muscle isoforms, to heterodimeric products of different tropomyosin genes, and to a portion of the TnT-TnI complex. Analyses of both myosin and tropomyosin proteins will also focus on specific regions implicated in various myopathies.
A third aim i s to focus X-ray crystallographic and computer analyses on selected alpha-helical coiled-coil segments from these and other proteins that contain specialized features such as alanine staggers, cavities, and trigger sequences. The goal of these studies is to provide a deeper understanding of how these factors influence the geometry, stability and flexibility of the molecules in relation to their function. Alpha-helical coiled coils have been shown to play a role in many oncogenic proteins. Here we have the opportunity to use knowledge gained from these studies of muscle protein alpha-helical coiled coils to help develop small molecule drugs aimed at targeting these structures. One example we focus on is a region of the human smooth-muscle myosin rod that is implicated in leukemia.
The overall aim of the project is to determine atomic structures of proteins critical for muscle contraction and its regulation. Mutations in these proteins are involved in diseases of the heart and other muscles, and have also been shown to cause some leukemias. A deeper understanding of the factors controlling the conformation and stability of these proteins - in particular the widespread alpha-helical coiled-coil motif - will be sought in order to help develop small molecule drugs that selectively target oncoproteins in diseased cells.
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