Journal of Cellular Biochemistry 83:99±110 (2001) Characterization of the Biologically Important Interaction Between Troponin C and the N-Terminal Region of Troponin I Sai-Ming Ngai 1,2 * and Robert S. Hodges 1,2 1 Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada 2 Medical Research Council of Canada Group in Protein Structure and Function, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Abstract The N-terminal regulatory region of Troponin I, residues 1±40 (TnI 1±40, regulatory peptide) has been shown to have a biologically important function in the interactions of troponin I and troponin C. Truncated analogs corresponding to shorter versions of the N-terminal region (1±30, 1±28, 1±26) were synthesized by solid-phase methodology. Our results indicate that residues 1±30 of TnI comprises the minimum sequence to retain full biological activity as measured in the acto-S1-TM ATPase assay. Binding of the TnI N-terminal regulatory peptides (TnI 1±30 and the N-terminal regulatory peptide (residues 1±40) labeled with the photoprobe benzoylbenzoyl group, BBRp) were studied by gel electrophoresis and photochemical cross-linking experiments under various conditions. Fluorescence titrations of TnI 1±30 were carried out with TnC mutants that carry a single tryptophan ¯uorescence probe in either the N- or C-domain (F105W, F105W/C domain (88±162), F29W and F29W/N domain (1±90)) (Fig. 1). Low Kd values (Kd < 10 7 M) were obtained for the interaction of F105W and F105W/C domain (88±162) with TnI 1±30. However, there was no observable change in ¯uorescence when the ¯uorescence probe was located at the N-domain of the TnC mutant (F29W and F29W/N domain (1±90)). These results show that the regulatory peptide binds strongly to the C- terminal domain of TnC. J. Cell. Biochem. 83: 99±110, 2001. ß 2001 Wiley-Liss, Inc. Key words: Troponin; TnI-TnC interaction; peptide The contraction of striated skeletal muscle results from the sliding motion of myosin along the actin ®laments whereby chemical energy in the form of ATP is used [Huxley and Hanson, 1954; Huxley and Niedergerke, 1954]. This mechanism is regulated by Ca 2 and requires the regulatory protein troponin and tropomyo- sin (TM) [Ebashi and Endo, 1968; Endo and Obinata, 1981]. Troponin is a complex of three proteins: troponin C (TnC), which binds Ca 2 ; troponin T (TnT), which interacts with tropo- myosin and anchors troponin to the thin ®lament, and troponin I (TnI), the inhibitory component capable of binding to actin ®lament and inhibiting the actomyosin ATPase activity [Leavis and Gergely, 1984; Zot and Potter, 1987; Da Silva et al., 1993; Grabarek et al., 1992; Tobacman, 1996; Miki et al., 1998]. The regions of TnI involved in interactions with TnC were ®rst identi®ed by Syska et al. [1976] as three cyanogen bromide fragments, CN4 (residues 96±116), CN5 (residues 1±21), and CF2 (residues 1±47) that bound to a TnC- Sepharose af®nity column. However, only the CN4 fragment containing residues 96±116, named the inhibitory peptide, was able to bind to actin-TM and inhibit the acto-S1-TM ATPase activity [Syska et al., 1976]. Although, it is very well documented that the TnI inhibitory region (residues 104±115) interacts with TnC [Talbot ß 2001 Wiley-Liss, Inc. DOI 10.1002/jcb.1212 Abbreviations used: TnC, troponin C; TnI, troponin I; Ip, TnI inhibitory peptide Ac-TnI (104± 115) amide; BBRp, N a- benzoylbenzoyl TnI (1±40 residues) amide; TM, tropomyo- sin; S1, myosin subfragment 1; acto-S1, actin and myosin subfragment 1; TFA, tri¯uoroacetic; DTT, dithiothreitol; Ac, acetylated N-terminus; Amide, amidated C-terminus. Grant sponsor: The Medical Research Council of Canada; Grant sponsor: The Alberta Heart and Stroke Foundation (to R.S.H); Grant sponsor: An Alberta Heritage Foundation for Medical Research studentship (to S.M.N). *Correspondence to: Sai-Ming Ngai, Department of Bio- chemistry, The Chiese University of Hong Kong, Hong Kong SAR, China. E-mail: smngai@cuhk.edu.hk Received 23 February 2001; Accepted 25 April 2001