Theoretical and Computational Chemistry

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



The cardiac troponin complex (cTn) is an important regulatory protein in heart contraction. Upon binding of Ca2+, cTn undergoes a conformational shift that allows the troponin I switch peptide (cTnISP) to be released from the actin filament and bind to the troponin C hydrophobic patch (cTnC). Mutations and modifications to this complex can change its sensitivity to Ca2+ and alter the energetics of the transition from the Ca2+-unbound, cTnISP-unbound form to the Ca2+-bound, cTnISP-bound form. We utilized targeted MD (TMD) to obtain a trajectory of this transition pathway, followed by umbrella sampling to estimate the free energy associated with the cTnISP-cTnCHP binding and the cTnCHP opening events for wild-type (WT) cTn. We were able to reproduce experimental values for the cTnISP-cTnCHP binding event and obtain cTnCHP 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 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 cTnCHP. For these same mutations, we observed no general trend in the effect on the cTnISP-cTnCHP 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.


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Supplementary material

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Supplemental Material
Table S1: Values for the collective variables. Figure S1. Interhelical angle throughout the 20ns and 40ns TMD simulations. Figure S2. Interhelical Angle vs. Interhelical Distance. Figure S3. Coverage of Collective Variables During Umbrella Sampling Simulations.