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 recombinan t chicken troponin C fragments. They were designed to assess the effec ts of domain isolation, N-helix, and D/E linker helix on stability and calcium affinity. Four of the fragments include the N-terminal regula tory 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 t he apo form as measured by Delta G(N-->U,25)degrees. Separation of the domains also decreases the stability. Differences in values of Delta G(N-->U,25)degrees derived from urea and guanidine hydrochloride studi es allowed an estimation of the electrostatic component of the free en ergy of unfolding. Our measurements provide the first quantitative est imate of the stability for the apo-C-domain (Delta G(N-->U,25)degrees = -1.8 kcal/mol) which was obtained using the interaction free energy formalism of Schellman. There is an inverse correlation between calciu m affinity, binding cooperativity, and stability for all of these homo logously 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 dom ain organization of TnC are necessarily involved in the fine-tuning of the affinity and cooperativity of calcium binding. Though not directl y involved in calcium coordination, these structural features are impo rtant for signal transmission by troponin C in the troponin complex.