EXTENSIVE INTERACTIONS BETWEEN TROPONIN-C AND TROPONIN-I - ZERO-LENGTH CROSS-LINKING OF TROPONIN-I AND ACETYLATED TROPONIN-C

Interactions between troponin C (TnC) and troponin I (TnI) play an imp ortant role in the Ca2+-dependent regulation of vertebrate striated mu scle 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-ter minal domain. Subsequently, on the basis of the results of zero-length cross-linking, we suggested that the inhibitory region of TnI also in teracts with the N-terminal, regulatory domain of TnC [Leszyk, J., Gra barek, Z., Gergely, J., and Collins, J. H. (1990) Biochemistry 29, 299 -304]. In the present study, we acetylated the epsilon-NH2 groups of t he 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-et hyl-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 group s in TnI. The cross-linked heterodimer (AcCxI) was cleaved with CNBr a nd proteases, and the resulting cross-linked peptides were separated b y HPLC and then sequenced. Our results show extensive cross-linking be tween AcTnC and TnI, involving both the N-terminal and C-terminal doma ins of TnC, as well as the N-terminal, C-terminal, and inhibitory regi ons of TnI.