Relationship between stability and function for isolated domains of troponin C.

Results of spectroscopic thermal and chemical denaturation studies and calcium binding studies are presented for a series of five recombinant chicken troponin C fragments. They were designed to assess the effects of domain isolation, N-helix, and D/E linker helix on stability and calcium affinity. Four of the fragments include the N-terminal regulatory domain and one the C-terminal domain. For the regulatory domain, deletion of the N-helix or the D/E linker decreases the stability of the apo form as measured by delta GN-->U,25. Separation of the domains also decreases the stability. Differences in values of delta GN-->U,25 derived from urea and guanidine hydrochloride studies allowed an estimation of the electrostatic component of the free energy of unfolding. Our measurements provide the first quantitative estimate of the stability for the apo-C-domain (delta GN-->U,25 = -1.8 kcal/mol) which was obtained using the interaction free energy formalism of Schellman. There is an inverse correlation between calcium affinity, binding cooperativity, and stability for all of these homologously structured fragments. The calcium affinity and cooperativity are highest for the unstructured C-domain and lowest for the N-domain which has the highest stability. In view of the direct effects on the folding stability of the apo-N-domain, the N-helix and the bilobed domain organization of TnC are necessarily involved in the fine-tuning of the affinity and cooperativity of calcium binding. Though not directly involved in calcium coordination, these structural features are important for signal transmission by troponin C in the troponin complex.