The Cardiac Ca 2+-Sensitive Regulatory Switch, a System in Dynamic Equilibrium

Abstract

The Ca 2+-sensitive regulatory switch of cardiac muscle is a paradigmatic example of protein assemblies that communicate ligand binding through allosteric change. The switch is a dimeric complex of troponin C (TnC), an allosteric sensor for Ca 2+, and troponin I (TnI), an allosteric reporter. Time-resolved equilibrium Förster resonance energy transfer (FRET) measurements suggest that the switch activates in two steps: a TnI-independent Ca 2+-priming step followed by TnI-dependent opening. To resolve the mechanistic role of TnI in activation we performed stopped-flow FRET measurements of activation after rapid addition of a lacking component (Ca 2+ or TnI) and deactivation after rapid chelation of Ca 2+. Time-resolved measurements, stopped-flow measurements, and Ca 2+-titration measurements were globally analyzed in terms of a new quantitative dynamic model of TnC-TnI allostery. The analysis provided a mesoscopic parameterization of distance changes, free energy changes, and transition rates among the accessible coarse-grained states of the system. The results reveal that 1), the Ca 2+-induced priming step, which precedes opening, is the rate-limiting step in activation; 2), closing is the rate-limiting step in de-activation; 3), TnI induces opening; 4), there is an incompletely deactivated population when regulatory Ca 2+ is not bound, which generates an accessory pathway of activation; and 5), there is incomplete activation by Ca 2+—when regulatory Ca 2+ is bound, a 3:2 mixture of dynamically interconverting open (active) and primed-closed (partially active) conformers is observed (15°C). Temperature-dependent stopped-flow FRET experiments provide a near complete thermokinetic parameterization of opening: the enthalpy change (Δ H = −33.4 kJ/mol), entropy change (Δ S = −0.110 kJ/mol/K), heat capacity change (Δ C p = −7.6 kJ/mol/K), the enthalpy of activation ( δ ‡ = 10.6 kJ/mol) and the effective barrier crossing attempt frequency ( ν adj = 1.8 × 10 4 s −1).