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.