Interactions of troponin I and its inhibitory fragment (residues 104-115) with troponin C and calmodulin

Abstract

Fluorescent probes have been used to study the interaction of troponin I and its inhibitory peptide TnIp with troponin C, calmodulin, and the proteolytic fragments of calmodulin. The probes used included the noncovalently bound ligand TNS and the covalently attached labels dansyl and AEDANS. The fluorescence intensity of TNS bound to troponin C, calmodulin, or the calmodulin fragments was greatly enhanced by the presence of TnIp. This effect was used to estimate the corresponding binding constants. It was found that TnIp is bound by the C-terminal half-molecule of calmodulin, TR2C, with an affinity comparable to that of intact calmodulin or troponin C, while the binding affinity of the N-terminal half-molecule, TR1C, was an order of magnitude less, suggesting that the TnIp-containing portion of troponin I combines with the C-terminal half of calmodulin or troponin C. The fluorescence properties of an AEDANS group linked to Cys-98 of troponin C were modified by interaction with troponin I or TnIp. The fluorescence properties of the same group linked to Cys-27 of wheat germ calmodulin were affected by TnI, but not TnIp. TnI had a small effect upon the fluorescence of a dansyl group linked to Met-25 of troponin C. TnIp also inhibited the tryptic hydrolysis of the midpoint of the central connecting strand of calmodulin and troponin C.(ABSTRACT TRUNCATED AT 250 WORDS)

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.…”

“…Several studies indicate that the TnI peptide interacts with the C-terminal domain of TnC (Weeks & Perry, 1978;Grabarek et al, 1981;Cachia et al, 1983;Drabikowski et al, 1985;Leszyk et al, 1987Leszyk et al, , 1988Lan et al, 1989). In the present study we use 'H NMR spectroscopy to compare the binding of TnIp to the synthetic noncovalent heterodimer comprised of site 111 and site IV peptides with the binding of TnIp to calcium-saturated s-TnC.…”

Functional recovery of troponin I in a Drosophila heldup mutant after a second site mutation.

“…A large number of reports have studied the molecular interaction between TnI and TnC (Horwitz et al, 1979;Lan et al, 1989;Leszyk et al, 1990;Miki, 1990;Wang et al, 1990;Farah et al, 1994;Guo et al, 1994;Kobayashi et al, 1994). In vitro experiments with purified proteins and also with synthetic peptides have established the interactions among several muscle proteins and the participating domains.…”

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).…”

“…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). This is in excellent agreement with our observation that Met 120 and Met 157, both at the outer edge of the hydrophobic pocket in the C-terminal domain of cTnC, are protected by the binding of cTnI peptide.…”