Elucidating the Structural Interaction between N- and C-Termini of Human Adult Cardiac Troponin T

Abstract

Troponin (Tn) is a ternary protein complex that imparts calcium regulation to striated muscle. This complex consists of cardiac Troponin I (cTnI, the inhibitory subunit), cardiac Troponin C (cTnC, the calcium binding subunit), and cardiac Troponin T (cTnT, the tropomyosin binding subunit). Previous studies have shown the Tn complex consists of two domains, an N-terminal extension of cTnT and the core domain (cTnI, cTnC, and the rest of cTnT). These studies primarily explored the troponin core. Here, we investigate the less well-known N-terminal extension in reconstituted thin filaments utilizing fluorescence resonance energy transfer (FRET). We aim to use FRET to determine the structural relationship between the N-terminal extension and the troponin core, as well as between the N- and C-termini of cTnT. We hypothesize the N-terminal extension may interact with the Tn core and/or the C-terminus of cTnT. To test this, donor and acceptor probes were placed on the N- and C-termini of cTnT and cTnC. Preliminary data indicates FRET has not occurred between IAEDANS labelled cTnT A2C and DABMI labelled cTnC G125C, suggesting the N-terminus may be further away than this probe pair is able to detect. Continuing work aims to probe additional sites on the N-terminal extension, troponin core, and C-terminus of cTnT, in order to determine the structure of the Tn complex. Additionally, we will utilize DDPM and 5-IAF acceptor probes with R0 values ranging from 30Å to 60Å to further probe these structures. These data will inform us of where the N-terminal extension is in relation to the troponin core and adjacent troponin complexes. Filling this gap in our understanding of the structure of the cTnT N-terminal extension is paramount to our understanding of the regulatory mechanism(s) of the cardiac thin filament.