| Troponin is a heterotrimeric complex that acts as a molecular switch on the thin filament of striated muscle, linking myosin-based contractile function with calcium signaling. The activation of the thin filament can be broadly described in three stages: blocked, closed, and open, each characterized by a tropomyosin position that permits or restricts myosin access. Troponin, particularly the troponin I subunit, undergoes large conformational changes to move tropomyosin through these activation states and permit the cyclical contraction and relaxation cycle of the heartbeat. However, many details of troponin's function remain uncertain.;The C-terminus of troponin I (TnI-C) is a roughly fifty-residue long domain believed to undergo a disorder-order transition during muscle relaxation (moving from a free, unbound conformation in the open state to a folded, bound conformation in the blocked state). It is believed that this transition progresses via a fly-casting mechanism, in which the disorder in TnI-C's open state allows it to locate its binding site on the thin filament quickly. TnI-C binding the thin filament may be the event that begins the sequence of conformational changes leading to the shifting of tropomyosin and inhibition of the thin filament, making this transition highly important in understanding muscle function at a molecular level. This transition is also believed to be dysregulated in hypertrophic cardiomyopathy, a genetic cardiac disease whose underlying molecular mechanism is poorly understood. Therefore, understanding the TnI-C fly-casting mechanism is important from both basic science and clinical perspectives.;This dissertation details our efforts to study the open state of TnI-C. The disordered state of a protein undergoing coupled binding and folding can define the kinetics and binding mechanism of the interaction. Here, we use a combined approach of single-molecule fluorescence and simulations to elucidate the conformation and dynamics of TnI-C in its disordered state, drawing upon a history of intrinsically disordered protein research that has informed many relevant techniques and approaches. We detail new methods for comparing simulations and experiments differing in timescales by orders of magnitude, and develop new analyses to evaluate intrinsically disordered regions, which have varying degrees of disorder throughout their sequences.;Finally, we present a picture of TnI-C in the open conformation of the solution-state troponin complex. TnI-C is a highly dynamic, extended domain, well poised for thin filament interaction. While disordered, TnI-C frequently samples three conformations, which are qualitatively similar to previous publications of contradictory TnI-C "structures". We show that even with these three clusters of conformations, TnI-C is globally rather homogeneous in its conformation, and that short molecular dynamics trajectories can probe its underlying dynamics. These analyses uncover extensive negatively correlated motion in the domain, driven in part by charge distribution, that is responsible for maintaining TnI-C in its extended conformation. This reliance on charge for TnI-C's conformation suggests a potential for alterations through targeted mutations or small molecules, both of which could be useful in the laboratory or as pharmaceutical targets for disease. We conclude by presenting potential directions for future research into this intrinsically disordered region, which could serve as a model protein for understanding coupled binding and folding as well as serve a medical purpose. |
