Dynamic Effects of Tropomyosin D230N Mutation and Fetal Troponin T on the Tropomyosin Overlap Region

Abstract

Introduction: Mutations in the cardiac thin filament cause changes in protein structure and dynamics. These alterations result in the complex tissue remodeling seen in patients with hypertrophic and dilated cardiomyopathies. Our group is investigating the effects of these mutations on the tropomyosin (Tm) head-to-tail overlap domain. In particular, we are investigating the alpha-Tm D230N mutation that causes a unique bimodal distribution of remodeling. We hypothesize that this is caused by the modulatory effects of the fetal form of cTnT (cTnT1) on overlap structure and function.Methods: Tm was modified to have one cysteine at residue 271 for both wildtype and D230N mutations, and was labeled with the FRET acceptor DDPM. cTnT1 or adult cardiac troponin T (cTnT3) were modified to have one cysteine at residue 100 and labeled with the FRET donor AEDANS. Labeled cTnT1 or cTnT3 were reconstituted into the troponin complex and combined with labeled Tm of either wildtype or D230N. Steady-state and lifetime data was collected.Results: The distance between the labeled sites in the wildtype complex increased with calcium activation. In contrast, the calcium activated state of the D230N complex resulted in a shorter measured distance. cTnT1 also resulted in a closer distance. cTnT1 in addition to D230N mutation resulted in a closer orientation compared D230N or cTnT1 alone. Differences in measured distances caused by the variants were more pronounced in the calcium activated state than in the low calcium state.Conclusion: The increased distance measured with wildtype complex supported the increased flexibility of Tm accompanying thin filament activation. The decreased distance measured at the Tm overlap supports the hypothesis that cTnT1 interacts with D230N in a manner that amplifies the effects of the mutation alone at the Tm overlap region.