Binding of Ca2+ to the regulatory domain of troponin C (TnC) in cardiac muscle initiates a series of protein conformational changes and modified protein−protein interactions that initiate contraction. Cardiac TnC contains two Ca2+ binding sites, with one site being naturally defunct. Previously, binding of Ca2+ to the functional site in the regulatory domain of TnC was shown to lead to a decrease in conformational entropy (TΔS) of 2 and 0.5 kcal mol-1 for the functional and nonfunctional sites, respectively, using 15N nuclear magnetic resonance (NMR) relaxation studies [Spyracopoulos, L., et al. (1998) Biochemistry 37, 18032−18044]. In this study, backbone dynamics of the Ca2+-free regulatory domain are investigated by backbone amide 15N relaxation measurements at eight temperatures from 5 to 45 °C. Analysis of the relaxation measurements yields an order parameter (S2) indicating the degree of spatial restriction for a backbone amide H−N vector. The temperature dependence of S2 allows estimation of the contribution to protein heat capacity from pico- to nanosecond time scale conformational fluctuations on a per residue basis. The average heat capacity contribution (Cp,j) from backbone conformational fluctuations for regions of secondary structure for the regulatory domain of cardiac apo-TnC is 6 cal mol-1 K-1. The average heat capacity for Ca2+ binding site 1 is larger than that for site 2 by 1.3 ± 0.8 cal mol-1 K-1, and likely represents a mechanism where differences in affinity between Ca2+ binding sites for EF hand proteins can be modulated.
|Translated title of the contribution||Temperature Dependence of Dynamics and Thermodynamics of the Regulatory Domain of Human Cardiac Troponin C|
|Pages (from-to)||12541 - 12551|
|Number of pages||11|
|Publication status||Published - Sep 2001|