Characterization of the biologically important interaction between troponin C and the N‐terminal region of troponin I

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 fluorescence 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 fluorescence when the fluorescence 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.

An NMR and spin label study of the effects of binding calcium and troponin I inhibitory peptide to cardiac troponin C

“…A number of other studies have also concluded that TnIp interacts primarily with regions in the C-terminal domain (Lan et al, 1989;Wang et al, 1990;Ngai & Hodges, 1992;Swenson & Frederickson, 1992;Ngai et al, 1994). Fluorescent probes reveal that, although intact sTnI is bound to both the N-and C-terminal domains of sTnC, the inhibitory fragments of sTnI (96-1 16 and 104-1 15) interact mainly with the C-terminal domain connecting strand of sTnC (Lan et al, 1989).…”

“…Numerous studies have suggested that sTnIp interacts with both the N-and C-terminal domains of sTnC (Kobayashi et al, 1991;Ngai & Hodges, 1992;Swenson & Frederickson, 1992;Farah et al, 1994). An explanation for these observations is that TnC, like CaM, folds over the peptide such that both domains make contact with the inhibitory peptide.…”

“…However, small-angle X-ray scattering experiments on TnC complexed with intact TnI and TnIp have shown that TnC is extended in both complexes (Blechner et al, 1992;Olah et al, 1994). Therefore, it seems more likely that TnIp can interact with both domains of TnC under appropriate conditions (Ngai & Hodges, 1992). It is now well known that the N-terminal region of TnI interacts with the C-terminal domain of TnC (Farah et al, 1994;Kobayashi et al, 1994;Krudy et al, 1994).…”

Calcium‐dependent protein–protein interactions induce changes in proximity relationships of Cys48 and Cys64 in chicken skeletal troponin I

“…Their data provided evidence in support of a significant function of a cTnT-binding domain in cTnI, and they suggested that cTnT binding to the N-terminus of cTnI is a negative regulator of cardiac activation. More recently, Ngai et al have characterized a minimum sequence of the N-terminus of TnI (residues 1-30) that retains full biological activity to bind the C-terminal domain of TnC [49], and showed that the N-and C-terminal domains of TnI act as a Ca 2+ -dependent switch for TnC [50]. Thus, the N-terminal region of TnI may participate in the calcium activation of muscle contraction in cardiac and skeletal muscle.…”

“…Based on several experimental approaches, such as crystallography [1][2][3], NMR [4][5][6], neutron scattering [7,8], chemical crosslinking [9-11] and fluorescence resonance energy transfer [12,13], a structural model of the TnC-TnI complex was proposed in which TnI winds around TnC in either a lefthanded manner (Model ÔLÕ) or a right-handed manner (Model ÔRÕ) [14]. According to these models, five adjoining segments of TnI, segment I-V, correspond to residues 3-33, [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][95][96][97][98][99][100][101][102][103][104][105][106][107][108][109][110][111][112][113][114] respectively. Segment…”