Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The most diverged region of the primary amino acid sequence between cardiac (cTnC) and fast skeletal troponin C is the N-terminal ten amino acids. We report here that major changes in the primary sequence of this region in cTnC had a minimal effect on the ability of the mutant proteins to recover maximal activity in TnC-extracted cardiac and fast skeletal muscle myofibrils. However, deletion of the N-terminal nine amino acids resulted in a 60% decrease in maximal Ca(2+)-dependent ATPase activity with only a small change in the pCa50 of activation. Deletion of the N-terminal peptide did not appear to appreciably affect the Ca(2+)-binding properties of cTnC, but it did alter the interaction with hydrophobic fluorescent probes. Thus, the presence but not the sequence, of the N-terminal extension is important for the maximal activity of cTnC. The N-terminal helix may function in a relatively non-specific manner to prevent unfavorable interactions between domains in cTnC or between cTnC and other troponin subunits.
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PMID:The presence but not the sequence of the N-terminal peptide in cardiac TnC is important for function. 803 94

Various thio-reactive bifunctional crosslinkers as well as 5,5'-dithiobis(2-nitrobenzoate)-mediated disulfide bond formation were used to crosslink troponin-C and troponin-I, the Ca(2+)-binding and inhibitory subunits of troponin, respectively. In all cases, substantial crosslinking was obtained when the reactions were carried out in the absence of Ca2+. No disulfide crosslinking occurred if either Cys98 of TnC, or Cys133 of TnI were blocked, indicating that these thiols are involved in the crosslinking. Troponin containing the disulfide crosslink is no longer capable of regulating actomyosin ATPase activity in a Ca(2+)-dependent manner. Our results suggest that the relative movement between the Cys98 region of TnC and the Cys133 region of TnI is required for the Ca(2+)-regulatory process in skeletal muscle.
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PMID:A disulfide crosslink between Cys98 of troponin-C and Cys133 of troponin-I abolishes the activity of rabbit skeletal troponin. 807 39

Troponin C has a 14-residue alpha-helix at the extreme amino terminus (the N-helix) which is absent in calmodulin. To learn the significance of this region in troponin C, residues 1-14 were deleted using site-directed mutagenesis. Analysis of the mutant troponin C (delta 14-TnC) showed that deletion of the N-helix did not alter the secondary structure of troponin C. Like wild type troponin C, it exhibited Ca(2+)-dependent conformational changes based on electrophoretic mobility and increases in alpha-helix content. The thermal stability of delta 14-TnC, however, was 20 degrees C lower than wild type troponin C in the presence or absence of divalent cations because of destabilization of the amino-terminal domain. To determine the functional consequences of the deletion, its ability to relieve troponin I and IT inhibition of the actomyosin ATPase was assayed. The results show that the mutant could relieve troponin I inhibition in the presence and absence of Ca2+ but could relieve troponin IT inhibition only to 45-50% of the wild type level, even at high concentrations. Also, the calcium affinity of the low affinity sites is reduced as evidence by the 2.4-2.8-fold increase in Ca2+ concentration required to achieve half-maximal activation of the MgATPase and calcium titration of the metal-induced conformation monitored by far UV circular dichroism measurements. In addition, the N-helix is required for the full conformational change to take place upon the binding of Ca2+, but not Mg2+, to the high affinity sites. The results indicate that the N-helix of troponin C is important for the stability of troponin C and may play a vital role in the Ca(2+)-switching mechanism.
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PMID:The effects of deletion of the amino-terminal helix on troponin C function and stability. 814 78

Previous studies showed that conversion of the first Ca2+ ligand in Ca(2+)-binding sites III and IV from Asp to Ala decreased the affinity of cardiac TnC (cTnC) for the thin filament. Here, the functional consequences of mutation of the second ligand in the Ca(2+)-binding sites of cTnC were determined. Equilibrium dialysis and Tyr fluorescence studies showed that conversion of the second Ca2+ ligand to Ala (Asp-67, site II; Asn-107, site III; and Asn-143, site IV) inactivated all three Ca(2+)-binding sites in the free protein. Ca2+ binding to the mutated site II was not recovered upon association with a troponin complex, and proteins with this mutation were unable to regulate Ca(2+)-dependent ATPase activity in TnC-extracted myofibrils. However, Ca2+ binding was recovered at the mutated sites III and IV under the same conditions. Sequential addition of active and inactive cTnC proteins in a myofibril ATPase assay suggested that that Mg2+ binding was not recovered and that the recovered Ca2+ affinity of the mutated sites III and IV was much lower than that of the wild type in that the Ca2+ concentrations required for apparent thin filament binding by proteins containing mutations at sites III and/or IV were significantly greater than that required for the wild-type protein.
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PMID:Differential recovery of Ca2+ binding activity in mutated EF-hands of cardiac troponin C. 822 52

Three subunits of rabbit skeletal muscle troponin were expressed in and purified from Escherichia coli. The procedures were optimized, and the reconstituted troponin complex is highly homogeneous, stable, and obtainable in large quantities, allowing us to conduct crystallization studies of the troponin complex. The three subunits expressed and purified are beta-TnT(N'-208), TnI(C64A, C133S), and the wild type TnC. beta-TnT(N'-208) is a 25 kDa fragment of beta-troponin T, which consists of 208 amino acids and lacks 58 residues in the N-terminal variable region. TnI(C64A, C133S) is a mutant troponin I, in which Cys-64 and Cys-133 are replaced by Ala and Ser, respectively. Each subunit was separately expressed in E. coli, purified by column chromatography including HPLC, and reassembled to form troponin complex. The reconstituted troponin complex was not distinguishable from authentic troponin prepared from rabbit skeletal muscle; the acto-S1 ATPase rate, as well as the superprecipitation, was calcium-sensitive. Small flat crystals up to 0.2 mm long have been reproducibly obtained in preliminary crystallization trials.
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PMID:Reconstitution of rabbit skeletal muscle troponin from the recombinant subunits all expressed in and purified from E. coli. 828 38

A TnI cDNA was cloned from rabbit fast skeletal muscle, and site-directed mutagenesis was applied to replace all the three cysteine residues, Cys-48 and Cys-64 by Ala and Cys-133 by Ser. The mutant and wild-type TnI were expressed in E. coli and purified to homogeneity. No significant functional differences were observed between the mutant and the authentic TnI in terms of the interactions with TnT and TnC, and the ability of the reassembled Tn complex to regulate the acto-S1 ATPase activity in a calcium-dependent manner. These findings suggest that none of the cysteine residues in TnI are essential for the function of this protein and can be replaced to obtain a non-oxidizable mutant TnI which is much easier to handle and suitable as an alternative to the authentic TnI for various purposes, such as crystallization of TnI and the whole Tn, and 1H NMR studies.
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PMID:E. coli expression and characterization of a mutant troponin I with the three cysteine residues substituted. 849 52

The two globular Ca(2+)-binding domains of troponin C are connected by a three-turn, exposed central helix. The requirements of this helical linker for regulatory function are not fully understood. In the present work we investigated the structural requirement of the linker using a series of insertion mutations that differ in predicted flexibility. TnCinrc has a nine-residue flexible random coil insert, TnCinpp has a nine-residue rigid polyproline insert (three turns), and TnCin alpha h has a seven-residue insert with high potential of forming alpha-helix. TnCinrc and TnCinpp were defective in the activation of the regulated actomyosin ATPase activity in the presence of Ca2+ when compared to wild type or TnCin alpha h, suggesting that altering the flexibility of the central helix impairs the regulatory function of troponin C. TnCin alpha h, TnCinrc, and TnCinpp had 87% +/- 3, 62% +/- 3, and 58% +/- 2 of the wild type activity, respectively (n = 6). All insertions in the central helix resulted in elongation of molecule compared to wild type TnC as determined by Stokes' radius. The Ca(2+)-affinity, the Ca(2+)-dependence of the actomyosin ATPase, and the stability of the insertion mutants were similar to wild type. Deletions of up to two turns of the central helix have little effect on troponin C function [Dobrowolski, Z., Xu, G-Q., & Hitchcock-DeGregori, S. E. (1991) J. Biol. Chem. 266, 5703-5710]. In another mutant (TnCd11) the entire central helix, 87KEDAKGKSEEE97, was deleted. With TnCd11, activation of the actomyosin ATPase activity in the presence of Ca2+ was normal, but inhibition in the absence of Ca2+ was less effective. Interaction of TnCd11 with TnI was altered. There was a 2-fold excess of TnCd11 in reconstituted Tn complex, consistent with another report [Babu, A., Rao, V. G, Su, H., & Gulati, J. (1993) J. Biol. Chem. 268, 19232-19238]. Our results suggest that the native length and structure of the central helix are optimal for normal regulatory function and that connectivity alone is insufficient for TnC function.
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PMID:Investigation of the structural requirements of the troponin C central helix for function. 852 54

Exposure of an N-terminal hydrophobic region in troponin C is thought to be important for the regulation of contraction in striated muscle. To test this hypothesis, single Cys residues were engineered at positions 45, 81, 84, or 85 in the N-terminal hydrophobic region of cardiac troponin C (cTnC) to provide specific sites for attachment of blocking groups. A synthetic peptide, Ac-Val-Arg-Ala-Ile-Gly-Lys-Leu-Ser-Ser, or biotin was coupled to these Cys residues, and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity (70-80% inhibited) resulted when the peptide was conjugated to Cys-81 in cTnC(C81), while a lesser degree of inhibition (10-25% inhibited) resulted from covalent modification of cTnC(C84) and cTnC(C85). Inhibition was not due to an altered affinity of the cTnC(C81)/peptide conjugate for the myofibrils, and the Ca2+ dependence of ATPase activity was essentially identical to the unmodified protein. Thus, a subregion of the N-terminal hydrophobic region in cTnC is sensitive to disruption, while other regions are less important or can adapt to rather bulky blocking groups. The data suggest that Ca(2+)-sensitizing drugs may bind to the N-terminal hydrophobic region on cTnC but not interfere with transmission of the Ca2+ signal.
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PMID:Covalent binding of peptides to the N-terminal hydrophobic region of cardiac troponin C has limited effects on function. 855 May 67

Skeletal muscle contraction is regulated by Ca2+ binding to troponin (Tn), a complex of three proteins attached to the actin-tropomyosin filaments. We have been investigating key interactions of the Ca(2+)-binding protein TnC and the inhibitory protein TnI. Previously, we used 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI, and found that the N-terminal, regulatory domain of TnC formed cross-links to the inhibitory region of TnI (Leszyk, J., Grabarek, Z., Gergely, J. and Collins, J.H. (1990) Biochemistry 29, 299-304). In the present study we have used EDC to form cross-links between TnC and a synthetic peptide, based on residues 104-115 of TnI, which mimics intact TnI in its ability to inhibit actomyosin ATPase activity. Prior to cross-linking, we acetylated the epsilon-amino groups of the nine lysine residues of TnC in order to prevent intramolecular cross-linking. Cross-linked TnC-peptide products were cleaved with CNBr and several proteinases. The resulting cross-linked peptides were purified by HPLC and characterized by amino-acid sequence analysis. Our results indicate that the TnI peptide interacted most strongly with two sites in TnC: Glu-60 and/or Glu-61 in the N-terminal domain, and acidic residue(s) in segment 84-94 of the linker region which connects the N- and C-terminal domains of TnC.
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PMID:Interaction of a troponin I inhibitory peptide with both domains of troponin C. 863 10

Activation of cardiac actomyosin ATPase requires the occupation of the single low-affinity Ca(2+)-binding site of troponin C (cTnC). Previously, we demonstrated pronounced differences between mammals and cold-water salmonid fish in the Ca2+ sensitivity of cardiac preparations, particularly in relation to temperature [Churcotte, C., Moyes, C. D., Baldwin, K., Bressler, B., & Tibbits, G. F. (1994) Am. J. Physiol. 267, R62-R70]. In this study, we examine the extent to which cTnC structure could account for the observed differences in myofibrillar Ca2+ sensitivity. Salmonid (Oncorhynchus mykiss) cTnC was cloned, sequenced, and expressed in Escherichia coli as a maltose-binding protein fusion. The coding region has 87% homology with human cTnC cDNA and differs in 13 of 161 amino acid residues from the human/bovine/porcine isoform. The sequence corresponding to the single regulatory Ca(2+)-binding site II is completely homologous to that of mammals. The protein expressed exhibits optical properties similar (circular dichroism, intrinsic fluorescence) to those of cTnC purified from salmonid (Salmo salar) and bovine ventricle. A single tryptophan residue was introduced into the inactive Ca(2+)-binding site I (ScTnC-FW27) to facilitate Ca2+ titration. The Ca(2+)-binding constant (K1/2 = 5.33 pCa units) was within the range reported for the low-affinity sites of mammalian cTnC. Although differences in TnC primary structure are striking, Ca2+ affinity of intact cardiac myofibrils is likely influenced by interactions with other troponin proteins.
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PMID:Cloning and expression of salmon cardiac troponin C: titration of the low-affinity Ca(2+)-binding site using a tryptophan mutant. 879 56


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