Conformation and Dynamics of the Troponin I C-Terminal Domain: Combining Single-Molecule and Computational Approaches for a Disordered Protein Region

Abstract

In recent years, single-molecule Förster resonance energy transfer (smFRET) has emerged as a critical and flexible tool in structural biology, particularly in the study of highly dynamic regions and molecular assemblies. The usefulness of smFRET can be further extended by combining it with computational approaches, marrying the coarse-grained experimental data with higher-resolution in silico calculations. Here we use smFRET to determine six pairwise distances within the intrinsically disordered C-terminal domain of the troponin I subunit (TnIC) of the cardiac troponin complex. We used published conflicting structures of TnIC as starting models for molecular dynamics simulations, which were validated through successful comparison with smFRET measurements before extracting information on conformational dynamics. We flnd that pairwise distances between residues fluctuate widely in silico, but simulations are generally in good agreement with longer time scale smFRET measurements after averaging across time. Finally, Monte Carlo simulations establish that the lower-energy conformers of TnIC are indeed varied, but that the highest-sampled clusters resemble the published, conflicting models. In this way, we flnd that the controversial structures are simply stabilized local minima of this dynamic region, and a population including all three would still be consistent with spectroscopic measurements. Taken together, the combined approaches described here allow us to critically evaluate existing models of TnIC, giving insight into the conformation and dynamics of TnIC’s disordered state prior to its probable disorder–order transition. Moreover, they provide a framework for combining computational and experimental methods with diflerent time scales for the study of disordered and dynamic protein states.

Order–Disorder Transitions in the Cardiac Troponin Complex

“…19; 49 However, this structure was immediately controversial, 16; 18; 50 and even its own authors noted that they were surprised at the number of salt bridges between their structure and its putative actin binding site given the known low affinity between them. 49 More recent work has indicated that TnI C is disordered in the open state, 16; 18; 50; 51 which calls the NMR structure into question. While the structure could still prove to be relevant to understanding the blocked state conformation, the thin filament was not included in experimental measurements and thus its potential to be a blocked state conformation is unclear.…”

“…18; 19; 22; 23; 37; 50; 53 The separate structural models were recently established as one conformational ensemble using an integrated single-molecule fluorescence and simulation approach, which showed that the published models represent clusters of conformers favored in the disordered state (Figure 3, bottom panel). 51 The conformational ensemble was clustered by mapping 3.5 Å contacts, which highlights helices as strong contacts along the i,i+3 diagonal. The helical propensities clustered by the contact maps match the helical propensities in published structures: a single N-terminal helix in Cluster 1 matching the 1J1E structure, 23 two short helices in areas highlighted in the 1VDJ structure, 19 and a single C-terminal helix similar to that proposed by NMR and crystallography.…”

“…37; 50 The shorter helical length in silico is likely due to a combination of stabilized secondary structure in published models and increased in silico flexibility from simulating the peptide in isolation. 51 …”

“…The heterogeneity in shorter-range contacts at the C-terminus indicates increased disorder in this area, which has the potential to assist a fast structural transition to the bound, blocked state through coupled binding and folding (discussed further below). 51 …”

“…21; 54 To confirm that a loss of Ca 2+ does not lead to dissociation of the switch peptide and IR from TnC N , we performed single-molecule Förster Resonant Energy Transfer (FRET) measurements on a solution-state troponin complex under high- and low- Ca 2+ conditions (methods and controls may be found in Metskas and Rhoades, 2015). 51 Donor and acceptor fluorophores were conjugated to TnI 152 (in the switch peptide) and TnC 35 (in TnC N ), and monitored for changes in FRET that would indicate dissociation of the interaction. The use of a stable solution-state troponin complex (without the presence of actin or tropomyosin) ensured that the switch peptide, IR, and TnI C were unbound from the thin filament, which is otherwise difficult to achieve as an isolated state.…”