Nuclear magnetic resonance study on rabbit skeletal troponin C: calcium-induced conformational change.
Rabbit skeletal muscle troponin C (TnC) was investigated by means of 1H NMR in the presence of dithiothreitol that prevents dimerization of the protein. Two-dimensional (2D) 1H NMR spectra were observed in order to assign resonances to specific amino acids. One-dimensional 1H NMR spectra were observed as a function of Ca2+ concentration. The Ca2+-induced spectral change is categorized into two types: type 1 corresponds to the conformational change of the C-terminal-half domain (Ca2+ high-affinity sites) and type 2 to that of the N-terminal-half domain (Ca2+ low-affinity sites). From the 2D NMR spectra and Ca2+ titration data, it was suggested that (1) amide protons of Gly-108, Ile-110, Gly-144, and Ile-146 are hydrogen-bonded when the C-terminal-half domain binds 2 mol of Ca2+ and (2) hydrogen bonds of Gly-108, Ile-110, Gly-144, and Ile-146 are destroyed or weakened when the C-terminal-half domain releases 2 mol of Ca2+. Nuclear Overhauser enhancement difference spectra as well as the Ca2+ titration data suggested that a hydrophobic cluster is formed in the C-terminal-half domain when the C-terminal-half domain binds 2 mol of Ca2+. A hydrophobic cluster exists in the N-terminal-half domain without regard to Ca2+ binding to the N-terminal-half domain. The spectra of Tyr-10 showed both types of spectral change during the Ca2+ titration. The results suggested that Tyr-10 of apo-TnC interacts with the C-terminal-half domain.
Department of Polymer Science, Faculty of Science, Hokkaido University, Sapporo, Japan.
S
S
Tsuda
Y
Y
Hasegawa
M
M
Yoshida
K
K
Yagi
K
K
Hikichi
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D004229:Dithiothreitol / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000032:analysis*; D011487:Protein Conformation; D011817:Rabbits; D014336:Troponin / Q000032:analysis*; D019209:Troponin C
D014336:Troponin; D019209:Troponin C; D002118:Calcium; D004229:Dithiothreitol