Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin

Tropomyosin (Tpm) is an actin-binding coiled-coil protein. In muscle, it regulates contractions in a troponin/Ca2+-dependent manner and controls the thin filament lengths at the pointed end. Due to its size and periodic structure, it is difficult to observe small local structural changes in the coiled coil caused by disease-related mutations. In this study, we designed 97-residue peptides, Tpm1.1(64-154) and Tpm3.12(65-155), focusing on the actin-binding period 3 of two muscle isoforms. Using these peptides, we evaluated the effects of cardiomyopathy mutations: I92T and V95A in Tpm1.1, and congenital myopathy mutations R91P and R91C in Tpm3.12. We introduced a cysteine at the N-terminus of each fragment to promote the formation of the coiled-coil structure by disulfide bonds. Dimerization of the designed peptides was confirmed by gel electrophoresis in the presence and absence of dithiothreitol. Using circular dichroism, we showed that all mutations decreased coiled coil stability, with Tpm3.12(65-155)R91P and Tpm1.1(64-154)I92T having the most drastic effects. Our experiments also indicated that adding the N-terminal cysteine increased coiled coil stability demonstrating that our design can serve as an effective tool in studying the coiled-coil fragments of various proteins.