Conformation of a troponin-I peptide bound to troponin-C as determined by 1H NMR.

Abstract

The Ca+2-mediated interaction between troponin-l (Tn-I) and troponin-C (Tn-C) in the thin filament of skeletal muscle is an important element in the regulation of the actomyosin ATPase. It has been proposed that the structural changes produced by Ca+2 binding result in the presentation of regions on the surface of Tn-C capable of binding Tn-I with high affinity (1,2,3). In particular, it appears that residues 84–1151 on Tn-C (which represent the N-terminal side of Ca+2-binding site III) interact with residues 97–117 of Tn-I (3,5). A synthetic analog of the inhibitory region of Tn-I, Nα-acetyl-Tn-l(104–115)amide, has been made by solid phase synthesis. This region represents the minimum sequence necessary for inhibition of actomyosin ATPase activity and is found to bind to Tn-C in a Ca+2-dependent fashion, with a binding constant of 1 x 105M-1 (6,7). The following body of work primarily addresses the question of secondary structure adopted by the Tn-I peptide when bound to Tn-C. In order to probe the structure of the bound Tn-I peptide, high field two-dimensional Nuclear Magnetic Resonance (NMR) techniques are used. Possible binding sites on the Tn-C molecule are also considered in light of recent findings from cross-linking studies between rabbit s-Tn-C, to which a maleimide cross-linker molecule has been attached at Cys98, and residues on whole Tn-I (8).