title: Ca2(+)-dependent interactions between the C-helix of troponin-C and troponin-I. Photocross-linking and fluorescence studies using a recombinant troponin-C.
abstract: We have used in vitro mutagenesis to synthesize in Escherichia coli a recombinant rabbit skeletal troponin-C (designated as TnC57) in which Cys-98 was replaced with leucine, and Ala-57 in the C-helix of the N-terminal domain was replaced with cysteine. TnC57 labeled with the bifunctional photocross-linker benzophenone-4-maleimide could be photocross-linked with troponin-I in both the binary complex with troponin-I and in the ternary complex with troponin-I and troponin-T. The fluorescence lifetime of TnC57 labeled with the probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine decreased from 13.2 +/- 0.1 to 11.8 +/- 0.1 ns when Ca2+ bound to the low affinity triggering sites. Complexation with either troponin-I or both troponin-I and troponin-T resulted in significant increases in this lifetime both in the absence and the presence of Ca2+. In either the binary or the ternary complex, this lifetime increased from 15.5 to 18.0 ns upon Ca2+ binding to the low affinity sites. Complementary acrylamide-quenching studies yielded results that are consistent with the fluorescence lifetime results. Our results show that the C-helix of troponin-C interacts with troponin-I, in confirmation of recent zero-length cross-linking results (Leszyk, J., Grabarek, Z., Gergely, J., and Collins, J.H. (1990) Biochemistry 29, 299-304). Moreover, they are in support of a model (Herzberg, O., Moult, J., and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644) in which the binding of Ca2+ to the triggering sites in the N-terminal domain of troponin-C results in the movement of the B- and C-helices away from the central helix, thereby exposing a putative hydrophobic binding site for troponin-I.
authors.affiliation: Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts General Hospital 02114.
authors.forename: Z Y
authors.initials: ZY
authors.lastname: Wang
authors.forename: S
authors.initials: S
authors.lastname: Sarkar
authors.forename: J
authors.initials: J
authors.lastname: Gergely
authors.forename: T
authors.initials: T
authors.lastname: Tao
mesh_terms: D000818:Animals; D001577:Benzophenones / Q000378:metabolism; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008301:Maleimides / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D009154:Mutation; D010777:Photochemistry; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
chemical_list: D001577:Benzophenones; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D008301:Maleimides; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; C046109:benzophenone-4-maleimide; D002118:Calcium