Umbrella Sampling Simulations Measure Switch Peptide Binding and Hydrophobic Patch Opening Free Energies in Cardiac Troponin.

The cardiac troponin (cTn) complex is an important regulatory protein in heart contraction. Upon binding of Ca 2+ , cTn undergoes a conformational shift that allows the troponin I switch peptide (cTnI SP ) to be released from the actin filament and bind to the troponin C hydrophobic patch (cTnC HP ). Mutations and modifications to this complex can change its sensitivity to Ca 2+ and alter the energetics of the transition from the Ca 2+ -unbound, cTnI SP -unbound form to the Ca 2+ -bound, cTnI SP -bound form. We utilized targeted molecular dynamics (TMD) to obtain a trajectory of this transition pathway, followed by umbrella sampling to estimate the free energy associated with the cTnI SP -cTnC HP binding and the cTnC HP opening events for wild-type (WT) cTn. We were able to reproduce experimental values for the cTnI SP -cTnC HP binding event and obtain cTnC HP opening free energies in agreement with previous computational measurements of smaller cTnC systems. This excellent agreement for WT cTn demonstrated the strength of computational methods in studying the dynamics and energetics of the cTn complex. We then introduced mutations to the cTn complex that cause cardiomyopathy or alter its Ca 2+ sensitivity and observed a general decrease in the free energy of opening the cTnC HP . For these same mutations, we observed no general trend in the effect on the cTnI SP -cTnC HP binding event. Our method sets the stage for future computational studies on this system that predict the consequences of yet uncharacterized mutations on cTn dynamics and energetics.