Evolutionary and physics-guided modulation of Ca²⁺ sensitivity in Cardiac troponin C

Context Precise tuning of Ca ²⁺ 2 + sensitivity is essential for cardiac contractility, requiring modulation of force generation without disrupting native regulatory control. Cardiac troponin C acts as the primary Ca ²⁺ 2 + sensor, where subtle perturbations at the regulatory EF-hand site II can influence thin-filament activation. Here, we present an integrated evolutionary and physics-guided modeling framework to identify mutations that modulate Ca ²⁺ 2 + responsiveness while preserving conserved allosteric architecture. The results support a mechanistic interpretation in which Ca ²⁺ 2 + sensitization can emerge through multiple dynamical mechanisms, including enhanced Ca ²⁺ 2 + –structure coupling, increased conformational plasticity, or improved Ca ²⁺ 2 + retention within the regulatory EF-hand. Methods Evolutionary constraints were inferred from vertebrate ortholog sequences using a Potts-model framework implemented in GREMLIN. Physics-based energetic screening was performed with PyRosetta using the ref2015 energy function, and molecular dynamics simulations were conducted using AMBER 2024 with the ff14SB force field, TIP3P solvent, Joung–Cheatham ion parameters, and a 12-6-4 Lennard–Jones Ca ²⁺ 2 + model. Trajectories were analyzed using cpptraj to quantify Ca ²⁺ 2 + –structure coupling and EF-hand dynamics.