Evolutionary and physics-guided modulation of Ca 2 + sensitivity in Cardiac troponin C

Precise tuning of Ca $_{}^{2+}$ sensitivity is essential for cardiac contractility, requiring modulation of force generation without disrupting native regulatory control. Cardiac troponin C acts as the primary Ca $_{}^{2+}$ 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 $_{}^{2+}$ responsiveness while preserving conserved allosteric architecture. The results support a mechanistic interpretation in which Ca $_{}^{2+}$ sensitization can emerge through multiple dynamical mechanisms, including enhanced Ca $_{}^{2+}$ -structure coupling, increased conformational plasticity, or improved Ca $_{}^{2+}$ retention within the regulatory EF-hand.

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 $_{}^{2+}$ model. Trajectories were analyzed using cpptraj to quantify Ca $_{}^{2+}$ -structure coupling and EF-hand dynamics.