Extensive interactions between troponins C and I. Zero-length cross-linking of troponin I and acetylated troponin C.
Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca(2+)-dependent regulation of vertebrate striated muscle contraction. Earlier studies have led to the proposal that the "inhibitory region" (residues 96-116) of TnI binds to an alpha-helical segment of TnC comprising residues 89-100 in the nonregulatory, C-terminal domain. Subsequently, on the basis of the results of zero-length cross-linking, we suggested that the inhibitory region of TnI also interacts with the N-terminal, regulatory domain of TnC [Leszyk, J., Grabarek, Z., Gergely, J., & Collins, J. H. (1990) Biochemistry 29, 299-304]. In the present study, we acetylated the epsilon-NH2 groups of the nine lysines of TnC in order to avoid complications which may arise from intramolecular cross-linking between NH2 and COOH groups of TnC. We then activated the COOH groups of acetylated TnC (AcTnC) with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysuccinimide. The activated AcTnC was combined with TnI, and zero-length cross-links were formed between COOH groups in AcTnC and lysine epsilon-NH2 groups in TnI. The cross-linked heterodimer (AcCxI) was cleaved with CNBr and proteases, and the resulting cross-linked peptides were separated by HPLC and then sequenced. Our results show extensive cross-linking between AcTnC and TnI, involving both the N-terminal and C-terminal domains of TnC, as well as the N-terminal, C-terminal, and inhibitory regions of TnI.
Kobayashi
T
T
Department of Biological Chemistry, School of Medicine, University of Maryland, Biotechnology Institute, Baltimore 21201, USA.
Grabarek
Z
Z
Gergely
J
J
Collins
J H
JH
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002234:Carbodiimides; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000378:metabolism; D003488:Cyanogen Bromide; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D011817:Rabbits; D012697:Serine Endopeptidases / Q000378:metabolism; D014336:Troponin / Q000737:chemistry / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D010455:Peptides; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; C036813:lysyl endopeptidase; D003488:Cyanogen Bromide; D002118:Calcium