Defining the structure and dynamics of the N-cTnI-N-cTnC interface in cardiac thin filaments.

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The cardiac thin filament (cTF) is a crucial regulator of cardiac muscle contraction. The interface between the N-lobe of TnC and the N-terminus of cTnI (N-cTnI) is known to be a key modulator of calcium binding kinetics. This region contains the serine 23/24 phosphorylation sites, a target of protein kinase A during beta adrenergic signaling. Phosphorylation of this region has shown to produce an increase in calcium dissociation rate in the cTF system. This increase in calcium dissociation rate results in improved diastolic performance to meet hemodynamic demand. The structural mechanism for this observation has yet to be investigated in cTF in-vitro systems. We hypothesize that the N-cTnI contributes to stabilizing the calcium atom in site II via interactions with cTnC and phosphorylation of the serine 23/24 results in the N-cTnI moving away from cTnC allowing for the observed increase in calcium dissociation rate. In order to investigate these proposed structural changes, we performed time-resolved Forster resonance energy transfer (TR-FRET) experiments between cTnI-A28C to cTnC-84C, cTnI-A17C to cTnC-84C, and cTnI-A9C to cTnC-84C in cTF to map N-cTnI with respect to cTnC under different biochemical conditions. We found that N-cTnI is more disordered in the absence of calcium and become more ordered and closer to cTnC upon the addition of calcium. Phosphorylation of the Serine 23/24 sites resulted in an increase in distance and increase in FWHM for the A28C and A17C sites. This increase in distance and disorder observed may explain the increase in calcium dissociation rate as the N-cTnI moves away from cTnC allowing for the rapid release of the calcium atom from cTnC. This hypothesis will be further investigated using molecular dynamics approaches in order to gain atomistic level resolution of specific N-cTnI-N-cTnC interactions.