Regulated actin-based cellular locomotion is a property of all eukaryotic cells that is taken to the extreme of perfection in striated muscles. A small number of component parts form an assembly that responds rapidly, cooperatively and transiently to signal molecules. We have high-resolution structures of the four major components of the machine, myosin, actin, tropomyosin and troponin, but not of higher order complexes. Therefore we lack a specific mechanistic knowledge of how signaling information is communicated to and within the contractile apparatus. Mutations in the genes encoding sarcomeric proteins are the cause of cardio- and skeletal myopathies, and many of these mutations are at the ends of signaling cascades (tropomyosin and troponin-T, for example). The focus of the proposed research is one of these proteins, tropomyosin, the major regulator of the actin filament in muscle and non-muscle cells. The actin filament is the universal binding partner of tropomyosin, and there are no specific models for how or where actin binds. Our major goal is to define the molecular basis of binding specificity and regulatory function. We also present tropomyosin as a model coiled-coil, and suggest that what we learn will provide insight into how other coiled-coil proteins bind their targets and how mutations cause disease. The four aims are:
Aim 1. Analysis of the molecular evolution of genes encoding tropomyosin. We will construct a phylogenetic tree, measure the rate of evolution of individual residues, and construct an ancestral tropomyosin sequence. We hypothesize that tropomyosin became "necessary" when there was a need for a more robust actin cytoskeleton than that found in amoebae.
Aim 2. Structural bioinformatics analysis of phylogenetic relationships and test of hypothesis: the most conserved amino acid residues of tropomyosin include those involved in binding the highly- conserved protein actin, and regulatory functions. Conserved residues will be mutated in rat tropomyosin, and the effect on function of a recombinant protein will be tested.
Aim 3. Identification of requirements for molecular recognition by tropomyosin in the actin cytoskeleton in the cellular model system, Schizosaccharomyces pombe. We will test the requirement of conserved residues of yeast tropomyosin for growth, cytoskeletal function, polarity, dynamics and formation of the contractile ring.
Aim 4. Prediction of molecular recognition sites in tropomyosin and actin using molecular dynamics and docking simulations. We will construct a molecular model of predicted actin- tropomyosin complex using computational methods.
The main health relevance of the proposed research is to understand the molecular basis of cardio- and skeletal-myopathy-causing mutations. A bioinformatics analysis may explain why certain invertebrate tropomyosins are highly allergenic. There is the potential to develop therapeutic peptides to treat diseases involving this class of proteins.
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