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 Ca2+-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 Tn C in which natural Cys-98 was replaced by Leu, and a single Cys residu e was introduced at position 12 (TnC12) or 89 (TnC89). Cys residues of mutant TnCs were derivatized with 4-maleimidobenzophenone and photocr oss-linked to TnI in binary TnC.TnI complexes. After digestion with CN Br or proteases, crosslinked peptides were purified and sequenced. TnC 12 cross-linked at or near TnI Met-134 in a region known to be sensiti ve not only to occupancy of the regulatory Ca2+-binding sites of TnC b ut also to the contractile state of the thin filament. TnC89 cross-lin ked to TnI(108-113) in the inhibitory region. Taken together with earl ier findings, these results indicate that in the Tnc TnI complex, both domains of TnC, as well as the linker region between them, make conta ct 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 Tn I inhibitory region.