Structure of the troponin complex. Implications of photocross-linking of troponin I to troponin C thiol mutants.
Ca2+ regulation of vertebrate-striated muscle contraction is initiated by conformational changes in the Ca(2+)-binding protein troponin C (TnC) and subsequent changes in the interaction of TnC with the inhibitory protein TnI. We have constructed mutants of rabbit skeletal muscle TnC in which natural Cys-98 was replaced by Leu, and a single Cys residue was introduced at position 12 (TnC12) or 89 (TnC89). Cys residues of mutant TnCs were derivatized with 4-maleimidobenzophenone and photocross-linked to TnI in binary TnC.TnI complexes. After digestion with CNBr or proteases, cross-linked peptides were purified and sequenced. TnC12 cross-linked at or near TnI Met-134 in a region known to be sensitive not only to occupancy of the regulatory Ca(2+)-binding sites of TnC but also to the contractile state of the thin filament. TnC89 cross-linked to TnI(108-113) in the inhibitory region. Taken together with earlier findings, these results indicate that in the TnC.TnI complex, both domains of TnC, as well as the linker region between them, make contact with the inhibitory region of TnI. Our data also indicate that the N- and C-terminal domains of TnC interact with opposite ends of the TnI inhibitory region.
Kobayashi
T
T
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland.
Tao
T
T
Gergely
J
J
Collins
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D003432:Cross-Linking Reagents; D008969:Molecular Sequence Data; D009154:Mutation; D010777:Photochemistry; D011487:Protein Conformation; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014336:Troponin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019209:Troponin C; D019210:Troponin I
D003432:Cross-Linking Reagents; D013438:Sulfhydryl Compounds; D014336:Troponin; D019209:Troponin C; D019210:Troponin I