Definition of surface-exposed epitopes on the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum.
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ABSTRACT:
SUBMITTER: Tunwell RE
PROVIDER: S-EPMC1151567 | biostudies-other | 1991 Oct
REPOSITORIES: biostudies-other
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Project description:Peptides have been synthesized representing parts of the transduction, phosphorylation, nucleotide-binding and hinge domains of the (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR), and corresponding to segments of all of the postulated short inter-membranous loops of the (Ca(2+)-Mg2+)-ATPase (residues 77-88, 277-287, 780-791, 808-818, 915-924 and 949-958). A number of antibodies raised to these peptides have been shown to bind to the ATPase, defining surface-exposed regions. Many of these are concentrated in the phosphorylation and nucleotide-binding domains, suggesting that these domains could be exposed on the top surface of the ATPase. The cytoplasmic location of the loop containing residues 808-818 was confirmed by the finding that proteinase K treatment of intact SR vesicles enhanced the binding of antibodies against this segment. These findings support the 10-alpha-helix model of the ATPase. These results also suggest that only inter-membranous loops larger than about 20 residues are likely to be detected by immunological methods in transmembranous proteins. Binding of anti-peptide antibodies to proteolytic fragments of the ATPase has been used to define the domain structure of the enzyme. Some of the anti-peptide antibodies have been characterized by studying their binding to sets of hexameric peptides synthesized on plastic pegs. A wide pattern of responses is observed, with a restricted range of epitopes being recognized by each anti-peptide antibody.
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Project description:The sarcoplasmic reticulum Ca²? ATPase (SERCA) is a membrane-bound pump that utilizes ATP to drive calcium ions from the myocyte cytosol against the higher calcium concentration in the sarcoplasmic reticulum. Conformational transitions associated with Ca²?-binding are important to its catalytic function. We have identified collective motions that partition SERCA crystallographic structures into multiple catalytically-distinct states using principal component analysis. Using Brownian dynamics simulations, we demonstrate the important contribution of surface-exposed, polar residues in the diffusional encounter of Ca²?. Molecular dynamics simulations indicate the role of Glu309 gating in binding Ca²?, as well as subsequent changes in the dynamics of SERCA's cytosolic domains. Together these data provide structural and dynamical insights into a multistep process involving Ca²? binding and catalytic transitions.
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Project description:Equilibrium fluorescence methods have been used to establish a model for Ca2+ binding to the (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding. The basic scheme proposed is: E2 <--> E1 <--> E1Ca <--> El'Ca <--> E1'Ca2. The E1 conformation of the ATPase initially has one high-affinity binding site for Ca2+ exposed to the cytoplasmic side of the sarcoplasmic reticulum, but in the E2 conformation this site is unable to bind Ca2+; Ca2+ does not bind to luminal sites on E2. The second, outer, Ca(2+)-binding site on the ATPase is formed after binding of Ca2+ to the first, inner, site on E1 and the E1Ca <--> E1'Ca conformation change. The pH- and Mg(2+)-dependence of the E2 <--> E1 equilibrium has been established after changes in the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole. It is proposed that Mg2+ from the cytoplasmic side of the sarcoplasmic reticulum can bind to the first Ca(2+)-binding site on both E1 and E2. It is proposed that the change in tryptophan fluorescence intensity after binding of Ca2+ follows from the E1Ca <--> E1'Ca change. The pH- and Mg(2+)-dependence of this change defines H(+)- and Mg(2+)-binding constants at the two Ca(2+)-binding sites. It is proposed that the change in tryptophan fluorescence observed on binding Mg2+ follows from binding at the second Ca(2+)-binding site. Effects of pH and Mg2+ on the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin are proposed to follow from binding to a site on the ATPase, the 'gating' site, which affects the affinity of the first Ca(2+)-binding site for Ca2+ and affects the rate of dissociation of Ca2+ from the ATPase.
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Project description:Stop-flow fluorescence and rapid-filtration methods have been used to establish the kinetics of Ca2+ binding to, and dissociation from, the (Ca(2+)-Mg2+)-ATPase of skeletal-muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding and dissociation rates. The kinetics have been interpreted in terms of the scheme: E2 E2<==>E1<==>E1Ca<==>E1'Ca<==>E1'Ca2. The kinetics of the E2<==>E1 E1 transition have been determined by measuring the rate of change of the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole after a pH jump or the addition of Ca2+ to the labelled ATPase in the presence of thapsigargin or thapsivillosin A. It has been shown that Mg2+ has a marked effect on Ca2+ dissociation at pH 7.2 and that changes in the tryptophan fluorescence of the ATPase follow the same time course as the dissociation of 45Ca2+. It is proposed that the effect of Mg2+ follows from binding to a 'gating' site, as detected by changes in the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin. The rate of dissociation of Ca2+ from the ATPase increases with increasing pH. The rate of dissociation of Ca2+ decreases with increasing Ca2+ concentration in the medium, with an apparent affinity for Ca2+ greater than that seen for the change in fluorescence amplitude. It is shown that this follows if the first, inner, Ca(2+)-binding site on the ATPase has a lower affinity for Ca2+ than the second, outer, site. Effects of H+ and Mg2+ on Ca2+ dissociation can be treated by the quasiequilibrium approach. Mg2+ and H+ also affect the rate of Ca2+ binding to the ATPase, and effects of H+ and Mg2+ on the E2<==>E1 equilibrium explain the results of experiments in which the concentrations of H+ and Mg2+ are jumped.
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Project description:The use of Porphyrin derivatives as photosensitizers in Photodynamic Therapy (PDT) was investigated by means of a molecular docking study. These molecules can bind to intracellular targets such as P-type CaCa(2+) ATPase of sarcoplasmic reticulum (SERCA1a). CAChe software was successfully employed for conducting the docking of Tetraphenylporphinesulfonate(TPPS), 5,10,15,20- Tetrakis (4-sulfonatophenyl) porphyrinato Iron(III) Chloride (FeTPPS) and 5,10,15,20-Tetrakis (4-sulfonatophenyl) porphyrinato Iron(III) nitrosyl Chloride (FeNOTPPS) with CaCa(2+) ATPase from sarcoplasmic reticulum of rabbit. The results show that FeNOTPPS forms the most stable complex with CaCa(2+) ATPase.
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