Cross-linking of residue 57 in the regulatory domain of a mutant rabbit skeletal muscle troponin C to the inhibitory region of troponin I.
Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca(2+)-dependent regulation of vertebrate striated muscle contraction. In the present study, we investigated the sites of interaction between the N-terminal regulatory domain of TnC and the inhibitory region (residues 96-116) of TnI, using a mutant rabbit skeletal TnC (designated as TnC57) that contains a single Cys at residue 57 in the C-helix. TnC57 was modified with the photoreactive cross-linker 4-maleimidobenzophenone (BP-Mal), and, after formation of a binary complex with TnI, cross-linking between the proteins was induced by photolysis. The resulting product was cleaved with CNBr and several proteases, and peptides containing cross-links were purified and subjected to amino acid sequencing. The results show that Cys-57 of TnC57 is cross-linked to the segment of TnI spanning residues 113-121. Previously, we showed that Cys-98 of TnC can be cross-linked via BP-Mal to TnI residues 103-110 (Leszyk, J., Collins, J.H., Leavis, P.C., and Tao, T. (1987) Biochemistry 26, 7042-7047). Taken together, these results demonstrate that both the C- and the N-terminal domains of TnC interact with the inhibitory region of TnI and are consistent with the hypothesis that, in a complex with TnI, TnC adopts a more compact conformation than in the crystal structure.
Kobayashi
T
T
Department of Biological Chemistry, School of Medicine and Medical Biotechnology, University of Maryland, Baltimore 21201.
Tao
T
T
Grabarek
Z
Z
Gergely
J
J
Collins
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D002918:Chymotrypsin / Q000494:pharmacology; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D004252:DNA Mutational Analysis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins; D012697:Serine Endopeptidases / Q000494:pharmacology; D014336:Troponin / Q000737:chemistry*; D019209:Troponin C; D019210:Troponin I
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C036813:lysyl endopeptidase; D003488:Cyanogen Bromide