Using Isothermal Titration Calorimetry to Characterize the Ca2+ Binding Properties of Cardiac Troponin C and Slow Skeletal-specific Troponin C in Zebrafish

Slow skeletal-specific TnC (ssTnC, tnnc1b), found only in teleosts, and cardiac troponin C (cTnC, tnnc1a) are both expressed in zebrafish (ZF) heart in a temperature- and chamber-specific pattern. The focus of this study is to determine the Ca2+ binding affinities (KCa) of the regulatory site in ZF cTnC and ssTnC, and the thermal stability of each protein. Isothermal titration calorimetry was used to determine the KCa of the N- terminal domain of ZF cTnC and ssTnC at 8°C, 18°C and 28°C. The results for both TnCs show an increase in KCa with increasing temperature. The melting temperature (Tm) of each protein was also determined from thermal melting curves. The results show that ZF cTnC is slightly more stable with a Tm of 2°C higher. The data obtained provide functional insight into the Ca2+ binding properties of ZF TnC. It also provides a better understanding of teleost TnC sub-functionalization for chamber-specific expression seen in ZF.