Structural Effects of Cardiac Troponin T R92L and Tropomyosin D230N Mutants in the Cardiac Thin Filament

Introduction/Methods: Inherited mutations in cardiac thin filament proteins lead to changes in protein structure and dynamics. This results in the pathological tissue remodeling seen in patients with hypertrophic (HCM) and dilated (DCM) cardiomyopathies. Our group is interested in the alpha-Tm D230N mutation known to cause DCM and the cardiac troponin T (cTnT) mutation R92L which causes HCM. To investigate effects of these mutations on the tropomyosin (Tm) head-to-tail overlap we used Forster Resonance Energy Transfer (FRET). For both wild-type and mutants, Tm was labeled at site 271 with DDPM, cTnT was labeled with IAEDANS at site 100. Labeled cTnT was reconstituted into troponin complex (Tn) and combined with Tm along with actin to form the reconstituted thin filament. Time-resolved data was acquired and analyzed.Results/Conclusions: Both R92L and D230N mutants result in a change in distance between labeled sites of cTnT and Tm when compared to wild-type. Transfer efficiency decreased from 0.78 to 0.66 upon addition of the D230N mutation, representing an increased distance between the labeled sites. However, the transfer efficiency increased from 0.78 to 0.80 in the presence of the R92L mutation, supporting a decreased distance between the labeled sites. This differential modulation of the distance may reflect alterations in the interaction between the cTnT and Tm proteins due to these mutations. This supports the hypothesis that these mutations which cause different clinical phenotypes are having opposite effects in the region of interest. Ongoing experiments utilizing site 60 of cTnT, and sites 262 and 279 of Tm will provide additional information about the similar and differential effects of these mutants on the Tm overlap region.