A FRET Investigation on the Effects of Tropomyosin D230N and Cardiac Troponin T R92L Mutants on the Tropomyosin Overlap Structure

Cardiac thin filament protein mutants cause changes in protein structure and dynamics. This results in the pathological tissue remodeling seen in patients with hypertrophic (HCM) and dilated (DCM) cardiomyopathies. We propose that two mutations, alpha-tropomyosin (Tm) D230N, which is known to cause DCM, and cardiac troponin T (cTnT) R92L, which causes HCM, differentially affect the structure of the Tm overlap. To investigate the effects of these mutations on this important domain, we used time resolved Forster Resonance Energy Transfer (FRET). Fully reconstituted thin filaments were labeled with probes measuring the distance between cTnT and Tm.Given that the baseline structure is still unknown in this region, the results inform the interactions of the five helices that contribute to the Tm overlap. The cTnT R92L and Tm D230N mutants result in opposite effects on measured distances at the overlap region when compared to wild-type. This occurred between residues 100 and 127 of cTnT and residues 271 and 279 of Tm. The effects are located at the C-terminus of Tm, near a conserved hydrophobic core and a cTnT binding region. These opposing effects on distances at the overlap reflect an altered primary interaction between cTnT and Tm. Ongoing experiments investigating the N-terminal portion of Tm will be informative about propagation of effects across the overlap domain.