Residues 48 and 82 at the N-terminal hydrophobic pocket of rabbit skeletalmuscle troponin-C photo-cross-link to met121 of troponin-I

It has been proposed [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644] , and confirmed by structural studies [Gagne et al. (1995) Not. Struct. Bio l. 2, 784-789], that the binding of Ca2+ to the triggering sites in troponi n-C (TnC) causes the opening of the N-terminal hydrophobic pocket bound by the B, C, and D helices. This conformational change is believed to provide an additional binding site for troponin-I (TnI) and to lead to further even ts in the Ca2+ regulation process. To answer the question of which part of TnI interacts with this hydrophobic patch of TnC, we constructed two TnC mu tants, each with a single cysteine, one at residue 48 between helices B and C and the other at residue 82 on the D helix. Each mutant was labeled with the photoactivatable cross-linker benzophenone-4-iodoacetamide, followed b y reconstitution and UV irradiation. Studies were made in the binary comple x composed of TnC and TnI, the ternary complex composed of TnC, TnI, and tr oponin-Tr (TnT), and the synthetic thin filament composed of troponin, trop omyosin, and F-actin. TnC-TnI photo-cross-linking was observed for both mut ants and for all three types of complexes. Although no Ca2+ dependence in t he photo-crosslinking was observed on the binary and ternary complexes, the extent of cross-linking was reduced in the absence vs the presence of Ca2 in the thin filament. TnI Metl21, five residues from the C-terminus of the inhibitory region, was identified as the cross-linking site for both TnC m utants using microsequencing and mass spectrometry following proteolysis. T hese results, obtained with intact TnC TnI complexes, indicate that the TnT segment containing Met121 is in close contact with the N-terminal hydropho bic patch of TnC, and that in the thin filament the segment containing this residue moves away slightly from the hydrophobic patch in the absence of C a2+, possibly triggering the translocation of the actin-binding region(s) o f TnI toward actin.