Abstract

Cardiac troponin T (cTnT) serves as the tropomyosin (Tm) binding subunit of the troponin complex and, when mutated, it often leads to pathologic cardiac remodeling observed in patients with hypertrophic and dilated cardiomyopathies. Part of the N-terminus of cTnT is responsible for positioning Tm along the actin groove and it immediately flanks the Tm overlap domain. Understanding the molecular mechanisms underlying these observations has been hindered by the absence of a high-resolution structure in the highly flexible N-terminal domain. A recently published cryo-electron-microscopy structure shows interactions between the N-terminus of cTnT, around amino acid 87, and the F-actin backbone; however, this interaction was not observed in calcium saturated conditions. Here, we employ single-donor dual-acceptor time-resolved fluorescence resonance energy transfer (TR-FRET) to probe the positioning of the cTnT N-terminal extension in fully reconstituted cardiac thin filaments (CTF). We hypothesize the N-terminal tail interacts with F-actin in the −Ca2+ condition aiding in stabilizing Tm in the blocked position. Conversely, we hypothesize that the that the N-terminal tail is “fly-casting” and not interacting with F-actin in +Ca2+ condition. To test this, a donor probe (IAEDANS) was placed on either the Cys-substituted residues N100C, N73C and M60C of cTnT and an acceptor probe (DABMI) was placed on the endogenous Cys-374 of actin. Preliminary data revealed two measurable discrete distances for all residues, one short and one long, indicating that FRET occurred with two actin monomers. Initial results suggest that the N-terminal tail is interacting with F-actin in high and low calcium conditions. Continuing work aims to probe additional sites on the N-terminus, specifically A20C and A2C. Resolving the structure and orientation of the N-terminal domain of cTnT is critical to understanding the regulatory mechanisms of the CTF.

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