Evolutionary and Physics-Guided Modulation of Ca2+ Sensitivity in Cardiac Troponin C

Calcium sensitivity in cardiac muscle must be precisely tuned to enhance systolic force generation without impairing diastolic relaxation. Here we present a physics- and evolution-guided strategy to achieve modest, physiologically relevant Ca ²⁺ sensitization of cardiac troponin C by targeting its sole regulatory EF-hand. Using Potts-model-based evolutionary constraints as a form of negative design, we identify mutations that preserve native allosteric architecture while excluding perturbations that artificially stabilize the Ca ²⁺ -bound state. Molecular dynamics simulations show that effective sensitization does not arise from increased Ca ²⁺ affinity or nonspecific loop softening, but from enhanced coupling between Ca ²⁺ motion and EF-hand conformational response in the bound-state. These results establish a physical mechanism for tunable Ca ²⁺ regulation and demonstrate how evolutionary constraints can be exploited to engineer regulatory proteins without compromising physiological control.