Temperature dependence of dynamics and thermodynamics of the regulatory domain of human cardiac troponin C

Binding of Ca2+ to the regulatory domain of troponin C (TnC) in cardiac mus cle initiates a series of protein conformational changes and modified prote in-protein interactions that initiate contraction. Cardiac TnC contains two Ca2+ binding sites, with one site being naturally defunct. Previously, bin ding of Ca2+ to the functional site in the regulatory domain of TnC was sho wn to lead to a decrease in conformational entropy (T DeltaS) of 2 and 0.5 kcat mol(-1) for the functional and nonfunctional sites, respectively, usin g N-15 nuclear magnetic resonance (NMR) relaxation studies [Spyracopoulos, L., et al. (1998) Biochemistry 37, 18032-18044]. In this study, backbone dy namics of the Ca2+-free regulatory domain are investigated by backbone an-d de N-15 relaxation measurements at eight temperatures from 5 to 45 degreesC . Analysis of the relaxation measurements yields an order parameter (S-2) i ndicating the degree of spatial restriction for a backbone amide H-N vector . The temperature dependence of S-2 allows estimation of the contribution t o protein heat capacity from pico- to nanosecond time scale conformational fluctuations on a per residue basis. The average heat capacity contribution (C-pj) from backbone conformational fluctuations for regions of secondary structure for the regulatory domain of cardiac apo-TnC is 6 cat mol(-1) K-1 . The average heat capacity for Ca2+ binding site I is larger than that for site 2 by 1.3 +/- 0.8 cat mol(-1) K-1, and likely represents a mechanism w here differences in affinity between Ca2+ binding sites for EF hand protein s can be modulated.