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

Slupsky, Carolyn M.; Shaw, Gary S.; Campbell, A. Patricia; Sykes, Brian D.

doi:10.1002/pro.5560011207

Cited by 17 publications

(13 citation statements)

References 61 publications

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“…4; Kinemages 1, 2). This result corresponds to the previous observation based on ' H chemical shift changes, that sTnIp, an inhibitory peptide corresponding to Gly 104-Arg 115 of sTnI, interacts with Phe and Ile residues in the C-terminal hydrophobic pocket of sTnC (Cachia et al, 1983;Slupsky et al, 1992). Alternatively, binding of cTnIp to a site other than the C-terminal hydrophobic pocket could result in a conformational change that leads to a decrease in solvent exposure for both Met residues 120 and 157.…”

Section: Discussionsupporting

confidence: 90%

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

et al. 1995

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The paramagnetic relaxation reagent, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-l-oxy (HyTEMPO), was used to probe the surface exposure of methionine residues of recombinant cardiac troponin C (cTnC) in the absence and presence of Ca2+ at the regulatory site (site 11), as well as in the presence of the troponin I inhibitory peptide (cTnIp). Methyl resonances of the 10 Met residues of cTnC were chosen as spectral probes because they are thought to play a role in both formation of the N-terminal hydrophobic pocket and in the binding of cTnIp. Proton longitudinal relaxation rates (R,'s) of the [13C-methyl] groups in [13C-methyl]Met-labeled cTnC(C35S) were determined using a T I two-dimensional heteronuclear single-and multiple-quantum coherence pulse sequence. Solvent-exposed Met residues exhibit increased relaxation rates from the paramagnetic effect of HyTEMPO. Relaxation rates in 2Ca2+-loaded and Ca2+-saturated cTnC, both in the presence and absence of HyTEMPO, permitted the topological mapping of the conformational changes induced by the binding of Ca2+ to site 11, the site responsible for triggering muscle contraction. Calcium binding at site I1 resulted in an increased exposure of Met residues 45 and 81 to the soluble spin label HyTEMPO. This result is consistent with an opening of the hydrophobic pocket in the N-terminal domain of cTnC upon binding Ca2+ at site 11. The binding of the inhibitory peptide cTnIp, corresponding to Asn 129 through Ile 149 of cTnI, to both 2Ca2+-loaded and Ca2+-saturated cTnC was shown to protect Met residues 120 and 157 from HyTEMPO as determined by a decrease in their measured R, values. These results suggest that in both the 2Ca2+-loaded and Ca2+-saturated forms of cTnC, cTnIp binds primarily to the C-terminal domain of cTnC.Keywords: calcium; isotope labeling; paramagnetic relaxation; spin label; troponin C; troponin I peptide Muscle contraction is regulated by the TnC component of the troponin complex. The troponin complex is made up of TnC, the Ca2+ regulatory subunit, TnI, the subunit that inhibits actomyosin ATPase activity, and TnT, the tropomyosin-binding

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Section: Discussionsupporting

confidence: 90%

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

et al. 1995

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“…The proposal that residues 116 to 131 of TnI binds the N domain of TnC further supports the binding of the inhibitory region along the central helix and into the C domain (Cachia et al, 1983(Cachia et al, , 1986Chandra et al, 1994;Dalgarno et al, 1982;Drabikowski et al, 1985;Grabarek et al, 1981;Howarth et al, 1995;Jha et al, 1996;Kobayashi et al, 1994, Krudy et al, 1994Lan et al, 1989;Leszyk et al, 1987Leszyk et al, , 1988Ngai et al, 1994;Slupsky et al, 1992;Swenson & Fredricksen, 1992;Tsuda et al, 1992;Wang et al, 1990;Weeks & Perry, 1978). Grabarek et al (1981), using a variety of proteolytic fragments of TnC, have indicated that the region comprising residues 89 to 100 of the central helix constitutes one of the two Ca 2 -dependent sites of interaction between TnI and TnC, based on the observations that only TnC fragments containing these residues are able to regulate the actomyosin ATPase activity when complexed with TnI and TnT, presumably by binding to the inhibitory region of TnI in the presence of calcium.…”

Section: Binding Of the Inhibitory Region To The Central Helix And C mentioning

confidence: 72%

“…Furthermore, the fact that the release curves for peptides TnI 96 ± 148A1 and TnI 96 ± 148A7 are similar to that of TnI 96 ± 115 indicates that the interaction between the inhibitory region and TnC has not changed. As previously reported, it is believed that the inhibitory region interacts with the C domain of TnC (Cachia et al, 1983(Cachia et al, , 1986Chandra et al, 1994;Dalgarno et al, 1982;Drabikowski et al, 1985;Grabarek et al, 1981;Howarth et al, 1995;Jha et al, 1996;Kobayashi et al, 1994Kobayashi et al, , 1996Krudy et al, 1994;Lan et al, 1989;Leszyk et al, 1987Leszyk et al, , 1988Ngai et al, 1994;Slupsky et al, 1992;Swenson & Fredricksen, 1992;Tsuda et al, 1992;Wang et al, 1990;Weeks & Perry, 1978) and that the N domain, upon binding calcium, exposes a new hydrophobic surface area (Gagne et al, 1994(Gagne et al, , 1995Herzberg et al, 1986;. With the above results indicating that TnI residues 116 to 131 interact with TnC in a hydrophobic manner, these results strongly suggest that it is these residues that are interacting with the N domain of TnC.…”

mentioning

confidence: 79%

Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+-dependent regulation of muscle contraction

Tripet

Eyk

Hodges

1997

Journal of Molecular Biology

198

231

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“…Since both domains of TnC interact with TnI, and in view of the sequence homology and similar 3D structures of TnC and CaM, it seems that the basic features of the complexes formed by these proteins may be similar. 'H-NMR (Campbell et al, 1991;Slupsky et al, 1992) and molecular modeling studies (Strynadka & James, 1990) suggest that this indeed is the case in so far as the hydrophobic pocket in each domain of TnC is the docking site for the segments of TnI. However, there are some important differences.…”

Section: Discussionmentioning

confidence: 95%

Properties of Troponin C Acetylated at Lysine Residues

Grabarek

Mabuchi²,

Gergely³

1995

Biochemistry

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We have studied the properties of rabbit skeletal troponin C (TnC) fully acetylated at its lysine residues (AcTnC). Acetylation causes a decrease in thermal stability of both domains of TnC in the absence of Ca2+. At 25 degrees C, the acetylated C-terminal domain of TnC is almost completely unfolded and the melting temperature of the N-terminal domain monitored by far-UV circular dichroism is decreased by 16.3 degrees C. In the presence of 1 mM CaCl2, no cooperative unfolding can be detected up to 90 degrees C for either TnC or AcTnC. At 25 degrees C, CD spectra show that AcTnC has a slightly lower alpha-helix content in the absence of Ca2+, but higher in the presence of Ca2+ as compared to unmodified TnC. Acetylation causes a 3.5-fold increase in affinity for Ca2+ at the low-affinity sites and a 2-fold decrease at the high-affinity sites. Polyacrylamide gel electrophoresis under nondissociating conditions (no SDS, no urea, pH 8.6) indicates that acetylation has little effect on the apparent affinity of TnC for troponin I; however, the binding of the acetylated peptides corresponding to the N-terminal domain of TnC to troponin I is significantly stronger than that of the unmodified peptides. Troponin T binding to AcTnC is significantly enhanced, the altered properties of the N-terminal domain being predominantly responsible for the increase. Titration of the ATPase activity of TnC-depleted myofibrils with AcTnC or native TnC indicates that acetylation increases TnC's affinity for myofibrils in the presence of Ca2+ approximately 6 times; at saturation the ATPase activity is the same for the two forms of TnC. The Ca2+ dependence of the ATPase activity of myofibrils containing AcTnC is shifted to lower Ca2+ concentrations, consistent with the higher Ca2+ affinity of AcTnC at the low-affinity sites. These data indicate that positively charged lysine side chains, especially those located in the N-terminal domain, modulate TnC's structural stability and interactions with Ca2+ and other troponin components.

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

Cited by 17 publications

References 61 publications

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

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

Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+-dependent regulation of muscle contraction

Properties of Troponin C Acetylated at Lysine Residues

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

Cited by 17 publications

References 61 publications

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

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

Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+-dependent regulation of muscle contraction

Properties of Troponin C Acetylated at Lysine Residues

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