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Kerry S. Smith - One of the best experts on this subject based on the ideXlab platform.
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allosteric regulation of lactobacillus plantarum xylulose 5 phosphate fructose 6 phosphate phosphoketolase xfp
Journal of Bacteriology, 2015Co-Authors: Katie Glenn, Kerry S. SmithAbstract:ABSTRACT Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate Cooperativity for all substrates (X5P, F6P, and P i ) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13: 657–663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate Cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate Cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. IMPORTANCE Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate Cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate Cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate Cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains.
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Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)
Journal of Bacteriology, 2015Co-Authors: Katie Glenn, Kerry S. SmithAbstract:Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate Cooperativity for all substrates (X5P, F6P, and P i ) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13: 657–663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate Cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate Cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. IMPORTANCE Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate Cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate Cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate Cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains.
Jm Jault - One of the best experts on this subject based on the ideXlab platform.
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Structural insight into the Cooperativity between catalytic and noncatalytic sites of F1-ATPase.
BBA - Biochimica et Biophysica Acta, 2004Co-Authors: P. Falson, A. Goffeau, M. Boutry, Jm JaultAbstract:F1-ATPase, the catalytic sector of Fo-F1 ATPases-ATPsynthases, displays an apparent negative Cooperativity for ATP hydrolysis at high ATP concentrations which involves noncatalytic and catalytic nucleotide binding sites. The molecular mechanism of such Cooperativity is currently unknown. To get further insights, we have investigated the structural consequences of the single mutation of two residues: Q173L in the alpha-subunit and Q170Y in the beta-subunit of the F1-ATPase of the yeast Schizosaccharomyces pombe. These residues are localized in or near the Walker-A motifs of each subunit and their mutation produces an opposite effect on the negative Cooperativity. The betaQ170 residue (M167 in beef heart) is located close to the binding site for the phosphate-Mg moiety of the nucleotide. Its replacement by tyrosine converts this site into a close state with increased affinity for the bound nucleotide and leads to an increase of negative Cooperativity. In contrast, the alphaQ173L mutation (Q172 in beef heart) abolishes negative Cooperativity due to the loss of two H-bonds: one stabilizing the nucleotide bound to the noncatalytic site and the other linking alphaQ173 to the adjacent betaT354, localized at the alpha(DP)-beta(TP) interface. The properties of these mutants suggest that negative Cooperativity occurs through interactions between neighbor alpha- and beta-subunits. Indeed, in the beef heart enzyme, (i) the alpha(DP)-beta(TP) interface is stabilized by a vicinal alphaR171-betaD352 salt bridge (ii) betaD352 and betaT354 belong to a short peptidic stretch close to betaY345, the aromatic group of which interacts with the adenine moiety of the nucleotide bound to the catalytic site. We therefore propose that the betaY345-betaT354 stretch (beef heart numbering) constitutes a short link that drives structural modifications from a noncatalytic site to the neighbor catalytic site in which, as a result, the affinity for ADP is modulated.F1-ATPase, the catalytic sector of Fo-F1 ATPases-ATPsynthases, displays an apparent negative Cooperativity for ATP hydrolysis at high ATP concentrations which involves noncatalytic and catalytic nucleotide binding sites. The molecular mechanism of such Cooperativity is currently unknown. To get further insights, we have investigated the structural consequences of the single mutation of two residues: Q173L in the alpha-subunit and Q170Y in the beta-subunit of the F1-ATPase of the yeast Schizosaccharomyces pombe. These residues are localized in or near the Walker-A motifs of each subunit and their mutation produces an opposite effect on the negative Cooperativity. The betaQ170 residue (M167 in beef heart) is located close to the binding site for the phosphate-Mg moiety of the nucleotide. Its replacement by tyrosine converts this site into a close state with increased affinity for the bound nucleotide and leads to an increase of negative Cooperativity. In contrast, the alphaQ173L mutation (Q172 in beef heart) abolishes negative Cooperativity due to the loss of two H-bonds: one stabilizing the nucleotide bound to the noncatalytic site and the other linking alphaQ173 to the adjacent betaT354, localized at the alpha(DP)-beta(TP) interface. The properties of these mutants suggest that negative Cooperativity occurs through interactions between neighbor alpha- and beta-subunits. Indeed, in the beef heart enzyme, (i) the alpha(DP)-beta(TP) interface is stabilized by a vicinal alphaR171-betaD352 salt bridge (ii) betaD352 and betaT354 belong to a short peptidic stretch close to betaY345, the aromatic group of which interacts with the adenine moiety of the nucleotide bound to the catalytic site. We therefore propose that the betaY345-betaT354 stretch (beef heart numbering) constitutes a short link that drives structural modifications from a noncatalytic site to the neighbor catalytic site in which, as a result, the affinity for ADP is modulated.
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Glutamine 170 to tyrosine substitution in yeast mitochondrial F1 beta-subunit increases catalytic site interaction with GDP and IDP and produces negative Cooperativity of GTP and ITP hydrolysis.
Journal of Biological Chemistry, 1993Co-Authors: Jm Jault, G. Divita, Ws Allison, A. DipietroAbstract:Glutamine 170 to tyrosine mutation in the beta-subunit from Schizosaccharomyces pombe mitochondrial F1 was found to increase both affinity for ADP, apparent negative Cooperativity of ATPase activity, and sensitivity to azide inhibition (Falson, P., Di Pietro, A., Jault, J.-M., Gautheron, D.C., and Boutry, M. (1989) Biochim. Biophys. Acta 975, 119-126). The mutation is shown here to increase the affinity for GDP, IDP, and guanosine 5'-(beta,gamma-imidotriphosphate), which are competitive inhibitors of GTPase and ITPase activities. Various fluorescence approaches also reveal an increased affinity of the catalytic site in mutant as compared with wild-type enzyme for GDP, IDP, and 2'(3')-N-methylanthraniloyl GDP. The mutation alters the maximal rates and pH dependence of GTPase and ITPase activities, whereas wild-type F1 exhibits single optima at pH 7.5-8.0. The pH activity profiles of the mutant enzyme for these substrates are biphasic, with optima at pH 8.5-9.0 and below 6.5. The mutation increases the sensitivity of GTPase and ITPase activities to azide inhibition, which increases with decreasing pH. At pH 6.0-7.0, an apparent negative Cooperativity is observed when mutant F1 hydrolyzes GTP or ITP, whereas the wild-type enzyme shows Michaelian kinetics. Addition of bicarbonate at pH 7.0 substantially stimulates GTP or ITP hydrolysis and abolishes the apparent negative Cooperativity by the mutant enzyme; on the contrary, the anion produces a slight inhibition of these activities catalyzed by wild-type F1. The overall results suggest that apparent negative Cooperativity can be observed with GTP or ITP hydrolysis provided that the release of the respective diphosphate is a rate-limiting step.Glutamine 170 to tyrosine mutation in the beta-subunit from Schizosaccharomyces pombe mitochondrial F1 was found to increase both affinity for ADP, apparent negative Cooperativity of ATPase activity, and sensitivity to azide inhibition (Falson, P., Di Pietro, A., Jault, J.-M., Gautheron, D.C., and Boutry, M. (1989) Biochim. Biophys. Acta 975, 119-126). The mutation is shown here to increase the affinity for GDP, IDP, and guanosine 5'-(beta,gamma-imidotriphosphate), which are competitive inhibitors of GTPase and ITPase activities. Various fluorescence approaches also reveal an increased affinity of the catalytic site in mutant as compared with wild-type enzyme for GDP, IDP, and 2'(3')-N-methylanthraniloyl GDP. The mutation alters the maximal rates and pH dependence of GTPase and ITPase activities, whereas wild-type F1 exhibits single optima at pH 7.5-8.0. The pH activity profiles of the mutant enzyme for these substrates are biphasic, with optima at pH 8.5-9.0 and below 6.5. The mutation increases the sensitivity of GTPase and ITPase activities to azide inhibition, which increases with decreasing pH. At pH 6.0-7.0, an apparent negative Cooperativity is observed when mutant F1 hydrolyzes GTP or ITP, whereas the wild-type enzyme shows Michaelian kinetics. Addition of bicarbonate at pH 7.0 substantially stimulates GTP or ITP hydrolysis and abolishes the apparent negative Cooperativity by the mutant enzyme; on the contrary, the anion produces a slight inhibition of these activities catalyzed by wild-type F1. The overall results suggest that apparent negative Cooperativity can be observed with GTP or ITP hydrolysis provided that the release of the respective diphosphate is a rate-limiting step.
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Alteration of apparent negative Cooperativity of ATPase activity by alpha-subunit glutamine 173 mutation in yeast mitochondrial F1. Correlation with impaired nucleotide interaction at a regulatory site.
Journal of Biological Chemistry, 1991Co-Authors: Jm Jault, P. Falson, A. Dipietro, Dc GautheronAbstract:The first described alpha-subunit mutation of yeast mitochondrial F1 has been recently identified as a single Gln173----Leu substitution in a strongly conserved sequence (Falson, P., Maffey, L., Conrath, K., and Boutry, M. (1991) J. Biol. Chem. 266, 287-293). This mutation is shown here to greatly modify the biphasic pattern of ATPase activity as a function of pH: (i) the shoulder observed at acidic pH is significantly increased; (ii) the main peak, at alkaline pH, is markedly lowered; (iii) the optimal pH is shifted from 8.8 to 7.7. The mutation lowers both apparent negative Cooperativity and sensitivity to azide inhibition which concomitantly increase when the assay pH decreases. Azide partial inhibition produces apparent negative Cooperativity which can be further abolished by bicarbonate. The mutation increases both activation energies determined from biphasic Arrhenius plots. The mutation decreases the inactivation rate by 5'-p-fluorosulfonylbenzoyladenosine and abolishes the protection by nucleotide binding at the adenine-specific regulatory site. On the contrary, it does not modify the reactivity of 5'-p-fluorosulfonylbenzoylguanosine at the less-selective catalytic site. In addition, partial inactivation by 5'-p-fluorosulfonylbenzoyladenosine, as opposed to 5'-p-fluorosulfonylbenzoylguanosine, produces apparent negative Cooperativity under conditions where unmodified-enzyme kinetics are noncooperative. The results show that alpha-Gln173 participates in nucleotide interaction at a regulatory site which controls the negative Cooperativity of F1-ATPase activity.The first described alpha-subunit mutation of yeast mitochondrial F1 has been recently identified as a single Gln173----Leu substitution in a strongly conserved sequence (Falson, P., Maffey, L., Conrath, K., and Boutry, M. (1991) J. Biol. Chem. 266, 287-293). This mutation is shown here to greatly modify the biphasic pattern of ATPase activity as a function of pH: (i) the shoulder observed at acidic pH is significantly increased; (ii) the main peak, at alkaline pH, is markedly lowered; (iii) the optimal pH is shifted from 8.8 to 7.7. The mutation lowers both apparent negative Cooperativity and sensitivity to azide inhibition which concomitantly increase when the assay pH decreases. Azide partial inhibition produces apparent negative Cooperativity which can be further abolished by bicarbonate. The mutation increases both activation energies determined from biphasic Arrhenius plots. The mutation decreases the inactivation rate by 5'-p-fluorosulfonylbenzoyladenosine and abolishes the protection by nucleotide binding at the adenine-specific regulatory site. On the contrary, it does not modify the reactivity of 5'-p-fluorosulfonylbenzoylguanosine at the less-selective catalytic site. In addition, partial inactivation by 5'-p-fluorosulfonylbenzoyladenosine, as opposed to 5'-p-fluorosulfonylbenzoylguanosine, produces apparent negative Cooperativity under conditions where unmodified-enzyme kinetics are noncooperative. The results show that alpha-Gln173 participates in nucleotide interaction at a regulatory site which controls the negative Cooperativity of F1-ATPase activity.
Katie Glenn - One of the best experts on this subject based on the ideXlab platform.
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allosteric regulation of lactobacillus plantarum xylulose 5 phosphate fructose 6 phosphate phosphoketolase xfp
Journal of Bacteriology, 2015Co-Authors: Katie Glenn, Kerry S. SmithAbstract:ABSTRACT Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate Cooperativity for all substrates (X5P, F6P, and P i ) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13: 657–663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate Cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate Cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. IMPORTANCE Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate Cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate Cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate Cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains.
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Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)
Journal of Bacteriology, 2015Co-Authors: Katie Glenn, Kerry S. SmithAbstract:Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate Cooperativity for all substrates (X5P, F6P, and P i ) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13: 657–663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate Cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate Cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. IMPORTANCE Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate Cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate Cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate Cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains.
Amar H Flood - One of the best experts on this subject based on the ideXlab platform.
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electrostatic and allosteric Cooperativity in ion pair binding a quantitative and coupled experiment theory study with aryl triazole ether macrocycles
Journal of the American Chemical Society, 2015Co-Authors: Bo Qiao, Arkajyoti Sengupta, Yun Liu, Kevin P Mcdonald, Maren Pink, Joseph R Anderson, Krishnan Raghavachari, Amar H FloodAbstract:Cooperative binding of ion pairs to receptors is crucial for the manipulation of salts, but a comprehensive understanding of Cooperativity has been elusive. To this end, we combine experiment and theory to quantify ion-pair binding and to separate allostery from electrostatics to understand their relative contributions. We designed aryl-triazole-ether macrocycles (MC) to be semiflexible, which allows ion pairs (NaX; X = anion) to make contact, and to be monocyclic to simplify analyses. A multiequilibrium model allows us to quantify, for the first time, the experimental Cooperativity, α, for the equilibrium MC·Na(+) + MC·X(-) ⇌ MC·NaX + MC, which is associated with contact ion-pair binding of NaI (α = 1300, ΔGα = -18 kJ mol(-1)) and NaClO4 (α = 400, ΔGα = -15 kJ mol(-1)) in 4:1 dichloromethane-acetonitrile. We used accurate energies from density functional theory to deconvolute how the electrostatic effects and the allosteric changes in receptor geometry individually contribute to Cooperativity. Computations, using a continuum solvation model (dichloromethane), show that allostery contributes ∼30% to overall positive Cooperativity. The calculated trend of electrostatic Cooperativity using pairs of spherical ions (NaCl > NaBr > NaI) correlates to experimental observations (NaI > NaClO4). We show that intrinsic ionic size, which dictates charge separation distance in contact ion pairs, controls electrostatic Cooperativity. This finding supports the design principle that semiflexible receptors can facilitate optimal electrostatic Cooperativity. While Coulomb's law predicts the size-dependent trend, it overestimates electrostatic Cooperativity; we suggest that binding of the individual anion and cation to their respective binding sites dilutes their effective charge. This comprehensive understanding is critical for rational designs of ion-pair receptors for the manipulation of salts.
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electrostatic and allosteric Cooperativity in ion pair binding a quantitative and coupled experiment theory study with aryl triazole ether macrocycles
Journal of the American Chemical Society, 2015Co-Authors: Bo Qiao, Arkajyoti Sengupta, Kevin P Mcdonald, Maren Pink, Joseph R Anderson, Krishnan Raghavachari, Amar H FloodAbstract:Cooperative binding of ion pairs to receptors is crucial for the manipulation of salts, but a comprehensive understanding of Cooperativity has been elusive. To this end, we combine experiment and theory to quantify ion-pair binding and to separate allostery from electrostatics to understand their relative contributions. We designed aryl–triazole–ether macrocycles (MC) to be semiflexible, which allows ion pairs (NaX; X = anion) to make contact, and to be monocyclic to simplify analyses. A multiequilibrium model allows us to quantify, for the first time, the experimental Cooperativity, α, for the equilibrium MC·Na+ + MC·X– ⇌ MC·NaX + MC, which is associated with contact ion-pair binding of NaI (α = 1300, ΔGα = −18 kJ mol–1) and NaClO4 (α = 400, ΔGα = −15 kJ mol–1) in 4:1 dichloromethane–acetonitrile. We used accurate energies from density functional theory to deconvolute how the electrostatic effects and the allosteric changes in receptor geometry individually contribute to Cooperativity. Computations, usin...
Larry S. Tobacman - One of the best experts on this subject based on the ideXlab platform.
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Cooperative Binding of Tropomyosin to Actin
Advances in Experimental Medicine and Biology, 2008Co-Authors: Larry S. TobacmanAbstract:Tropomyosin molecules attach to the thin filament conjointly rather than separately, in a pattern indicating very high Cooperativity. The equilibrium process drawing tropomyosins together on the actin filament can be measured by application of a linear lattice model to binding isotherm data and hypotheses on the mechanism of Cooperativity can be tested. Each end of tropomyosin overlaps and attaches to the end of a neighboring tropomyosin, facilitating the formation of continuous tropomyosin strands, without gaps between neighboring molecules along the thin filament. Interestingly, the overlap complexes vary greatly in size and composition among tropomyosin isoforms, despite consistently cooperative binding to actin. Also, the tendency of tropomyosin to bind to actin cooperatively rather than randomly does not correlate with the strength of end-to-end binding. By implication, tropomyosin’s actin-binding Cooperativity likely involves effects on the actin filament, as well as direct interactions between adjacent tropomyosins.
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Analysis of troponin-tropomyosin binding to actin. Troponin does not promote interactions between tropomyosin molecules.
Journal of Biological Chemistry, 1992Co-Authors: Laura E Hill, Carol A. Butters, John P. Mehegan, Larry S. TobacmanAbstract:Abstract The binding of tropomyosin to actin and troponin-tropomyosin to actin was analyzed according to a linear lattice model which quantifies two parameters: Ko, the affinity of the ligand for an isolated site on the actin filament, and gamma, the fold increase in affinity when binding is contiguous to an occupied site (Cooperativity). Tropomyosin-actin binding is very cooperative (gamma = 90-137). Troponin strengthens tropomyosin-actin binding greatly but, surprisingly, does so solely by an 80-130-fold increase in Ko, while Cooperativity actually decreases. Additionally, troponin complexes containing TnT subunits with deletions of either amino acids 1-69 (troponin70-259) or 1-158 (troponin159-259) were examined. Deletion of amino acids 1-69 had only small effects on Ko and y, despite this peptide's location spanning the joint between adjacent tropomyosins. Ca2+ reduced Ko by half for both troponin and troponin70-159 and had no detectable effect on Cooperativity. Troponin159-259 had much weaker effects on tropomyosin-actin binding than did troponin70-259 and had no effect at all in the presence of Ca2+. This suggests the importance of Ca(2+)-insensitive interactions between tropomyosin and troponin T residues 70-159. Cooperativity was slightly lower for troponin159-259 than tropomyosin alone, suggesting that the globular head region of troponin affects tropomyosin-tropomyosin interactions along the thin filament.
