Structural and Functional Characterization of the TNT1 Domain of Cardiac Troponin T

Abstract

Familial Hypertrophic Cardiomyopathy (FHC) is a primary cardiac muscle disorder and a common cause of sudden cardiac death among young people in the field. The majority of disease-causing mutations in the thin filament protein hcTnT are found within the TNT1 domain. This domain has not been crystallized and its structural details are poorly defined, limiting our ability to understand the mechanism of disease for these mutations. A highly charged region is found at the C-terminal end of TNT1 (158-RREEEENRR-166) in which this highly alpha helical domain may unwind to create a flexible hinge that is necessary for normal function. We aim to determine the structural details and function of this region using SDSL-EPR and regulated in vitro motility (R-IVM) assays. The purpose of our R-IVM experiments is two-fold: to functionally analyze our spin labeled proteins and to gain insight into the function of TNT1 in the presence of cysteine substitutions. R-IVM data shows a progressive increase in the severity of the functional effects of cysteine substitution and spin labeling across the putative hinge region, suggesting that this region is dynamically important and may be making critical interactions with other components of the sarcomere. CW-EPR spectra of spin labeled hcTnT in Troponin ternary complexes show an increase in spin label mobility from residue 153 to 157 and 177, consistent with a decrease in alpha helical character across the putative hinge region. Preliminary doubly labeled CW-EPR experiments show that interspin distance between hcTnT residues 157 and 177 exceed 25A. Interspin distance measurements using doubly labeled hcTnT will further elucidate the secondary and tertiary structure of this region. Additional spin label pairs are currently being investigated using both CW-EPR and DEER techniques to determine the structural details of this important region.