Interactions of troponin subunits: free energy of binary and ternary complexes

Abstract

We have determined the free energy of formation of the binary complexes formed between skeletal troponin C and troponin T (TnC .cntdot. TnT) and between troponin T and troponin I (TnT .cntdot. TnI). This was accomplished by using TnC fluorescently modified at Cys-98 with N-(iodoacetyl)-N''-(5-sulfo-1-naphthyl)ethylenediamine for the first complex and TnI labeled at Cys-133 with the same probe for the other complex. The free energy of the ternary complex formed between troponin C and the binary complex TnT .cntdot. TnI [TnC .cntdot. (TnT .cntdot. TnI)] was also measured by monitoring the emission of 5-(iodoacetamido)eosin attached to Cys-133 of the troponin I in TnT .cntdot. TnI. The free energies were -9.0 kcal .cntdot. mol-1 for Tnc .cntdot. TnT, -9.2 kcal .cntdot. mol-1 for TnT .cntdot. TnI, and -8.7 kcal .cntdot. mol-1 for TnC .cntdot. (TnT .cntdot. TnI). In the presence of Mg2+ the free energies of TnC .cntdot. TnT and TnC .cntdot. (TnT .cntdot. TnI) were -10.3 and -10.9 kcal .cntdot. mol-1, respectively; in the presence of Ca2+ the corresponding free energies were -10.6 and -13.5 kcal .cntdot. mol-1. Mg2+ and Ca2+ had negligible effect on the free energy of TnT .cntdot. TnI. From these results the free energies of the formation of troponin from the three subunits were found to be -16.8 kcal .cntdot. mol-1, -18.9 kcal .cntdot. mol-1, and -21.6 kcal .cntdot. mol-1 in the presence of EGTA, Mg2+, and Ca2+, respectively. Most of the free energy decrease caused by Ca2+ binding to the Ca2+-specific sites is derived from stabilization of the TnI-TnC linkage. The free energy coupling for the formation of troponin from the three binary complexes was positive and large, indicating that the average of the binary interactions become relatively destabilized when the binary complexes were incorporated into the three-subunit troponin structure. The destabilization of subunit-subunit interactions may facilitate Ca2+-modulated interactions of the subunits with actin and tropomyosin which occur during the contractile cycle.