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

The cardiac troponin (cTn) complex is an important regulatory protein in heart contraction. Upon binding of Ca²⁺, 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 Ca²⁺ and alter the energetics of the transition from the Ca²⁺-unbound, cTnI_SP-unbound form to the Ca²⁺-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 Ca²⁺ 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.