Calcium dependence of the distance between Cys-98 of troponin C and Cys-133 of troponin I in the ternary troponin complex. Resonance energy transfer measurements

Abstract

We have used resonance energy transfer to study the spatial relationship between Cys-98 of rabbit skeletal troponin C and Cys-133 of rabbit skeletal troponin I in the reconstituted ternary troponin complex. The donor was introduced by labeling either troponin C or troponin I with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, while the acceptor was introduced by labeling either protein with N-[4-(dimethylamino)phenyl-4'-azophenyl]maleimide. The extent of energy transfer was determined by measuring the quenching of the donor fluorescence decay. The results indicate first that the distance between these two sites is not fixed, suggesting that the protein regions involved possess considerable segmental flexibility. Second, the mean distance between the two sites is dependent on the metal-binding state of troponin C, being 39.1 A when none of the metal-binding sites are occupied, 41.0 A when Mg2+ ions bind at the high-affinity sites, and 35.5 A when Ca2+ ions bind to the low-affinity sites. Neither the magnitude of the distances nor the trend of change with metal ions differs greatly when the locations of the probes are switched or when steady-state fluorometry was used to determine the transfer efficiency. Since the low-affinity sites have been implicated as the physiological triggering sites, our findings suggest that one of the key events in Ca2+ activation of skeletal muscle contraction is a approximately 5-A decrease in the distance between the Cys-98 region of troponin C and the Cys-133 region of troponin I.

Functional and structural similarities between the inhibitory region of troponin I coded by exon VII and the calmodulin-binding regulatory region of the catalytic subunit of phosphorylase kinase.

“…Although actin was found to neither stimulate nor inhibit the phosphotransferase activity of the y subunit alone (data not shown), we did find that actin inhibited both y-CaM and y-TnC with either phosphorylase b or tetradecapeptide as (25) and the y subunit of phosphorylase kinase (9) from rabbit fast skeletal muscle. The upper line for TnI represents the entire "inhibitory peptide" derived from CNBr cleavage (26,27).…”

“…The homologous region between the two proteins shown at the bottom of Fig. 1 does not have as clearly a defined function in TnI as the inhibitory peptide shown above it, although in the presence of Ca2+ this region of TnI, specifically Cys-133, is reportedly found closer to TnC (25) and further from actin (42). The homology in these two regions of the y subunit and TnI becomes more suggestive when one notes that every identical residue in the sequences shown in Fig.…”

A 1H NMR study of a ternary peptide complex that mimics the interaction between troponin C and troponin I

“…TnIp has been shown to interact with the C-terminal domain of TnC (Leavis et al, 1978;Weeks & Perry, 1978;Chong & Hodges, 1981;Grabarek et al, 1981;Wang & Cheung, 1984;Tao et al, 1986Tao et al, , 1989Leszyk et al, 1987;Lan et al, 1989). Here, we present a 'H NMR comparison of TnIp binding to s-TnC and TnIp binding to an SCIII/SCIV heterodimer.…”

“…Detailed NOE experiments should reveal where the site of TnIp binding is if peptide-protein NOES can be observed. Several reports suggest that the binding of the TnI peptide is to the N-terminal portion of site I11 (Leavis et al, 1978;Weeks & Perry, 1978;Chong & Hodges, 1981;Grabarek et al, 1981;Wang & Cheung, 1984;Tao et al, 1986Tao et al, , 1989Leszyk et al, 1987;Lan et al, 1989). Certainly, this region is highly conserved in s-TnC and CaM and is fairly acidic (Fig.…”

Mapping Subdomains in the C-terminal Region of Troponin I Involved in Its Binding to Troponin C and to Thin Filament

“…In the presence of calcium, the inhibitory region and the C terminus of TnI disconnect from the actin and bind to the N-and C-terminal regions of TnC, releasing the inhibition of the actomyosin ATPase activity (10,47,48). This work shows that the C-terminal region participates in various TnI functions: (i) the region between the inhibitory region and residue 148 is involved in TnC binding; (ii) the region between residues 98 and 129 is involved in modulating the affinity of TnC for calcium; (iii) the region between residues 166 and 182 is involved in binding to thin filament and is important for the inhibition of the actomyosin ATPase activity by Tn complex; and (iv) the region between the inhibitory region and residue 156 is important for the inhibition of the actomyosin ATPase activity by TnI alone.…”