Project summary/abstract: The goal of this proposal is to uncover how tropomyosin (Tpm) isoforms regulate actin filament function and how Tpm mutations cause muscle diseases such as cardiomyopathy, nemaline myopathy, and congenital myopathy. Progress in this area has been stalled by the inability to obtain physiologically relevant Tpm isoforms. Tpm isoforms play non-overlapping roles, and to function properly and self-associate head-to-tail along actin filaments, they must carry native post-translational modifications (PTMs) and be free of extra amino acids from purification tags. Traditional E. coli and yeast expression systems are not adequate in this case. In a major breakthrough, I have developed a system to express Tpm isoforms in human cells that overcomes these obstacles. In contrast to previous methods, my expressed Tpm isoforms (1) are N-terminally acetylated, (2) do not contain extra tag amino acids at the N- or C- termini, and (3) form natural heterodimers with other Tpm isoforms. In aim 1, I will optimize this expression method, including transfection efficiency and protein yields, so that it can be readily adopted by other scientists. I will further characterize the native state of each Tpm isoform, including the identification of natural heterodimer pairs and PTMs. In aim 2, I will address what are the biochemical differences among Tpm isoforms that determine their specialized cellular roles. I will focus on each isoform’s (or heterodimer’s) interactions with actin, and two regulatory proteins that must bind Tpm for their functions: tropomodulin (Tmod, a pointed end capping protein) and leiomodin (Lmod, a filament nucleator). In aim 3, I will study point mutations in Tpm responsible for muscle diseases, and address how these mutations disrupted the biochemical properties of Tpm, including its interactions with actin filaments, Tmod and Lmod. This knowledge should inform treatment strategies. The success of my aims depends mainly on the ability to express native Tpm isoforms, which I demonstrate in preliminary studies. My novel expression system uniquely positions me to address long-standing questions about Tpm function, and can be readily adopted by researchers wishing to study proteins in their native state. Accomplishment of the aims should have a far-reaching impact, providing both a biochemical understanding of Tpm isoform-specific cellular roles and information regarding heart and muscle disease mutations. This research will also serve to further my training in the fields of biochemistry, physiology, and molecular biophysics.