Kinetic Modeling Shows that the Apparent Rate of Troponin Dissociation from Myofibrils Probes the B-state to C-state Equilibrium of the Thin Filament

Abstract

The apparent rate of troponin (Tn) dissociation from myofibrils has been used as a method to study thin filament regulation. The rate is dependent upon calcium and strong crossbridges and supports the 3-state model for thin filament regulation (Swartz et al., 2007, J. Mol. Biol. 361:420). Tn dissociation rate is extremely slow so it is not intuitive that such a slow process probes thin filament regulation. To address this, we developed a simple two step kinetic model for Tn dissociation rate, as measured by labeled Tn exchange in myofibrils, and simulated the progress of labeled Tn incorporation into myofibrils and the intensity ratio of the non-overlap to overlap region. TnI's regulatory domain switches from actin-tropomyosin to TnC in the first step and TnT dissociates from actin-tropomyosin in the second step. Step 1 is the transition of the thin filament from the B-state to the C-state, is calcium dependent, and is several orders of magnitude faster than the forward reaction of step 2. By integrating the dimensionless rate equations of the above kinetic model, we have computed the time course of each of the various components. In our numerical simulations, the forward rate constant for step 1 was varied from 2 - 500 s-1 to simulate the pCa 9.0, B-state to pCa 4.0, C-state range. The progress curves for labeled Tn exchange into myofibrils and the derived intensity ratio, assuming a fixed exchange rate in the overlap region, well explain the observed intensity ratio progress curves for labeled Tn exchange into myofibrils. These simulations and experimental observations show that the apparent rate of Tn dissociation probes the B-state to C-state equilibrium of the myofibrillar thin filament. Supported by NIH-HLB.