CHARACTERIZATION OF ZERO-LENGTH CROSS-LINKS BETWEEN RABBIT SKELETAL-MUSCLE TROPONIN-C AND TROPONIN-I - EVIDENCE FOR DIRECT INTERACTION BETWEEN THE INHIBITORY REGION OF TROPONIN-I AND THE NH2-TERMINAL, REGULATORY DOMAIN OF TROPONIN-C

Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca2+-dependent regulation of vertebrate striated muscle contraction. Previous attempts to elucidate the molecular details of TnC-TnI interactions, mainly involving chemically modified proteins or fragments thereof, have led to the widely accepted idea that the “inhibitory region” (residues 96–116) of TnI binds to an α-helical segment of TnC comprising residues 89–100 in the nonregulatory, COOH-terminal domain. In an attempt to identify other possible physiologically important interactions between these proteins, l-ethyl-3-[3-(di-methylamino)propyl]carbodiimide (EDC) was used to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and Tnl. TnC was activated with EDC and N-hydroxysuccinimide (NHS) and then mixed with an equimolar amount of TnI [Grabarek, Z., & Gergely, J. (1988) Biophys. J. 53, 392a]. The resulting cross-linked TnC✕I was cleaved with cyanogen bromide, trypsin, and Staphylococcus aureus V8 protease (SAP). Cross-linked peptides were purified by reverse-phase HPLC and characterized by sequence analysis. The results indicated that residues from the regulatory Ca2+-binding site II in the NH2-terminal domain of TnC (residues 46–78) formed cross-links with TnI segments spanning residues 92–167. The most highly cross-linked residues in TnI were Lys-105 and Lys-107, located in the inhibitory region. These results yield the first evidence for an interaction between the N-terminal domain of TnC and the inhibitory region of Tnl. © 1990, American Chemical Society. All rights reserved.