Conformation of the Troponin I C-Terminal Domain in Silico and in vitro: A Consideration of Dynamics in Comparing Simulation and Experiment

Abstract

The troponin complex acts as a molecular switch in striated muscle cells to regulate myosin attachment to and isomerization on actin filaments in response to changes in calcium concentration. Transitions between the inactive and active states of the thin filament require extensive domain movements and binding exchanges involving the C-terminal domain of cardiac troponin I (TnI-C), believed to be intrinsically disordered in the high-Calcium state [1]. Mutations in TnI-C are associated with hypertrophic cardiomyopathy, highlighting the importance of this domain in regulating cardiac contraction; however, the conformational flexibility of the domain has delayed its characterization compared with the rest of the troponin complex.Here, we use single molecule Forster resonance energy transfer (smFRET) to probe the global conformation of TnI-C in the high-Calcium state, capitalizing on the technique's ability to analyze heterogeneous populations. We compare six pairwise distances within TnI-C to outputs from molecular dynamics simulations to gain insight into conformational sampling at finer detail and a faster timescale than experimental measurements allow. We find that simulations are in good agreement with smFRET measurements, but only after simulations are averaged over time. Simulations rarely, if ever, sample an “ideal” conformation matching all experimental measurements simultaneously; this finding highlights the importance of considering timescale when combining simulations and experimental measurements. Using our combined in silico and in vitro approach, we can predict areas of helical propensity and cluster potential global domain conformations in the unbound state, potentially providing mechanistic insight into the coupled binding and folding of this region during muscle relaxation.1. Julien, O., Mercier, P., Allen, C.N., et al. (2011) Is there nascent structure in the intrinsically disordered region of troponin I? Proteins 79: 1240-1250.