Site Directed Mutagenesis of Tropomyosin

Hitchcock-Degregori, Sarah

Abstract

The long term objective of the research is to understand the mechanism of tropomyosin function in thin filament linked regulation. Tropomyosin assembles with troponin along filaments and together they are primarily responsible for calcium dependent regulation of contraction in striated muscles. Tropomyosin allows the thin filament to function as a cooperative unit. The proposal focuses on a set of hypotheses concerning the basic structural features of tropomyosin required for function. From a broader perspective, tropomyosin is the best studied example of a coiled-coil fibrous protein. Molecular genetics offers an unprecedented opportunity to investigate structure-function relationships in tropomyosin in particular and coiled coils in general. The methodological approach will be site-directed mutagenesis of tropomyosin cDNAs and overexpression of mutant and wildtype cDNAs in E. coli for production of protein to be analyzed using biochemical, biophysical and cellular methods.
The Specific Aims are: 1. To obtain a fully functional recombinant tropomyosin that retains the both actin binding and regulatory properties of tropomyosin isolated from muscle; the role of post-translational acetylation in tropomyosin function. 2. To determine the role of the conserved amino terminus in the function of muscle tropomyosin; is it an actin binding site? 3. To determine if the internal periodic repeats of tropomyosin, their length and sequence are important for actin binding and regulation. 4. To determine the functional significance of tropomyosin exon structure. 5. To evaluate the parameters of the heptapeptide repeat that are determinants of short range and long range communication, stability and cooperativity in tropomyosin.