Structure, dynamics, and thermodynamics of the structural domain of troponin C in complex with the regulatory peptide 1-40 of troponin I

The structure of the calcium-saturated C-domain of skeletal troponin C (CTn C) in complex with a regulatory peptide comprising residues 1-40 (Rp40) of troponin I (Tnl) was determined using nuclear magnetic resonance (NMR) spec troscopy. The solution structure determined by NMR is similar to the struct ure of the C-domain from intact TnC in complex with TnI(1-47) determined by X-ray crystallography [Vassylyev, D. G., Takeda, S., Wakatsuki, S., Maeda, K., and Maeda, Y. (1998) Proc. Nad. Acad. Sci. U.S.A. 95, 4847-4852]. Chan ges in the dynamic properties of CTnC . 2Ca(2+) induced by Rp40 binding wer e investigated using backbone amide N-15 NMR relaxation measurements. Analy sis of NMR relaxation data allows for extraction of motional order paramete rs on a per residue basis, from which the contribution of changes in picose cond to nanosecond time scale motions to the conformational entropy associa ted with complex formation can be estimated. The results indicate that bind ing of Rp40 decreases backbone flexibility in CTnC, particularly at the end of the C-terminal helix. The backbone conformational entropy change (-T De ltaS) associated with binding of Rp40 to CTnC . 2Ca(2+) determined from N-1 5 relaxation data is 9.6 +/- 0.7 kcal mol(-1) at 30 degreesC. However, esti mation of thermodynamic quantities using a structural approach [Lavigne, P. , Bagu, J. R., Boyko, R., Willard, L., Holmes, C. F., and Sykes, B. D. (200 0) Protein Sci. 9, 252-264] reveals that the change in solvation entropy up on complex formation is dominant and overcomes the thermodynamic "cost" ass ociated with "stiffening" of the protein backbone upon Rp40 binding. Additi onally, backbone amide 15N relaxation data measured at different concentrat ions of CTnC . 2Ca(2+). Rp40 reveal that the complex dimerizes in solution. Fitting of the apparent global rotational correlation time as a function o f concentration to a monomer-dimer equilibrium yields a dimerization consta nt of similar to8.3 mM.