title: Temperature dependence of dynamics and thermodynamics of the regulatory domain of human cardiac troponin C.
abstract: Binding of Ca(2+) 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 Ca(2+) binding sites, with one site being naturally defunct. Previously, binding of Ca(2+) to the functional site in the regulatory domain of TnC was shown to lead to a decrease in conformational entropy (TDeltaS) of 2 and 0.5 kcal mol(-1) for the functional and nonfunctional sites, respectively, using (15)N nuclear magnetic resonance (NMR) relaxation studies [Spyracopoulos, L., et al. (1998) Biochemistry 37, 18032-18044]. In this study, backbone dynamics of the Ca(2+)-free regulatory domain are investigated by backbone amide (15)N relaxation measurements at eight temperatures from 5 to 45 degrees C. Analysis of the relaxation measurements yields an order parameter (S(2)) indicating the degree of spatial restriction for a backbone amide H-N vector. The temperature dependence of S(2) 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 (C(p,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 Ca(2+) 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 Ca(2+) binding sites for EF hand proteins can be modulated.
authors.affiliation: Department of Biochemistry, The Protein Engineering Network of Centers of Excellence, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada. leo.spyracopoulos@ualberta.ca
authors.forename: L
authors.initials: L
authors.lastname: Spyracopoulos
authors.forename: P
authors.initials: P
authors.lastname: Lavigne
authors.forename: M P
authors.initials: MP
authors.lastname: Crump
authors.forename: S M
authors.initials: SM
authors.lastname: Gagné
authors.forename: C M
authors.initials: CM
authors.lastname: Kay
authors.forename: B D
authors.initials: BD
authors.lastname: Sykes
mesh_terms: D000577:Amides / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002942:Circular Dichroism; D006358:Hot Temperature; D006801:Humans; D008956:Models, Chemical; D009206:Myocardium / Q000737:chemistry*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature; D013816:Thermodynamics; D019209:Troponin C / Q000737:chemistry*
chemical_list: D000577:Amides; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D019209:Troponin C; D002118:Calcium