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F A Quiocho

Publications and source records attributed to F A Quiocho.

At least 109 records · Page 6Linked to original sources
PubMed1988

Sulfate-binding protein dislikes protonated oxyacids. A molecular explanation.

We have determined the effect of pH on the binding affinities of the conjugate bases of four different tetrahedral oxyacids to the sulfate-binding protein. The equilibrium dissociation constants of the binding of sulfate (Kd = 0.12 microM) and selenate (Kd = 5 microM) were found to be pH independent over the range pH 5 to pH 8.1, whereas chromate binding exhibited a pH dependence that is approximately attributable to the pK2 of the chromic acid. Phosphate was bound with an affinity five orders of magnitude weaker than that of sulfate. In light of the highly refined 2 A structure of the complex of the sulfate-binding protein with sulfate, and considering the protonation state and net charge of the various oxyacids, we conclude that the pH dependence of chromate binding and the extremely low affinity of phosphate are attributable mainly to a lack of hydrogen bond acceptors in the binding site. These studies demonstrate that the sulfate-binding site is stringently designed to bind tightly tetrahedral, fully ionized, oxyacid dianions. The presence of a donatable proton on the ligand reduces binding energy by approximately 7 kcal/mol.

Bacterial Proteins↗
PubMed1988

Sugar and signal-transducer binding sites of the Escherichia coli galactose chemoreceptor protein.

D-galactose-binding (or chemoreceptor) protein of Escherichia coli serves as an initial component for both chemotaxis towards galactose and glucose and high-affinity active transport of the two sugars. Well-refined x-ray structures of the liganded forms of the wild-type and a mutant protein isolated from a strain defective in chemotaxis but fully competent in transport have provided a molecular view of the sugar-binding site and of a site for interacting with the Trg transmembrane signal transducer. The geometry of the sugar-binding site, located in the cleft between the two lobes of the bilobate protein, is novel in that it is designed for tight binding and sequestering of either the alpha or beta anomer of the D-stereoisomer of the 4-epimers galactose and glucose. Binding specificity and affinity are conferred primarily by polar planar side-chain residues that form intricate networks of cooperative and bidentate hydrogen bonds with the sugar substrates, and secondarily by aromatic residues that sandwich the pyranose ring. Each of the pairs of anomeric hydroxyls and epimeric hydroxyls is recognized by a distinct Asp residue. The site for interaction with the transducer is about 18 A from the sugar-binding site. Mutation of Gly74 to Asp at this site, concomitant with considerable changes in the local ordered water structures, contributes to the lack of productive interaction with the transmembrane signal transducer.

Bacterial Proteins↗
PubMed1988

Preliminary X-ray analysis of crystals of murine adenosine deaminase.

We have obtained single crystals of a cloned mammalian adenosine deaminase (Mr = 41,000), a key enzyme in purine degradation and in normal development of the immune system, that are suitable for high-resolution structural analysis. The crystals belong to the space group C2 with unit cell parameters a = 101.68 A (1 A = 0.1 nm), b = 94.38 A, c = 85.51 A, and beta = 96.54 degrees. The asymmetric unit contains two enzyme molecules.

Adenosine Deaminase↗
PubMed1988

The 2 A resolution structure of the sulfate-binding protein involved in active transport in Salmonella typhimurium.

The crystal structure of the liganded form of the sulfate-binding protein, an initial receptor for active transport of sulfate in Salmonella typhimurium, has been solved and refined at 2.0 A resolution (1 A = 0.1 nm). The final model, which consists of 2422 non-hydrogen atoms, one sulfate substrate and 143 water molecules, yields a crystallographic R-factor of 14.0% for 16,959 reflections between 8 and 2 A. The structure deviates from ideal bond lengths and angle distances by 0.015 A and 0.037 A, respectively. The protein is ellipsoid with overall dimensions of 35 A x 35 A x 65 A and consists of two similar globular domains. The two domains are linked by three distinct peptide segments, which though widely separated in the amino acid sequence, are in close proximity in the tertiary structure. As these connecting segments are located near the periphery of the molecule, they further serve as the base or a "boundary" of the deep cleft formed between the two domains. Despite the unusual interdomain connectivity, both domains have similar supersecondary structure consisting of a central five-stranded beta-pleated sheet sandwiched by alpha-helices on either side. The arrangement of the two domains gives rise to the ellipsoidal shape and to the cleft between the two domains wherein the sulfate substrate is found and completely engulfed. A discovery of considerable importance is that the sulfate substrate is tightly held in place primarily by seven hydrogen bonds, five of which are donated by main-chain peptide NH groups, another by a serine hydroxyl and the last by the indole NH moiety of a tryptophan side-chain; there are no positively charged residues, nor cations, nor water molecules within van der Waals' distance to the sulfate dianion. All the main-chain peptide units associated with the sulfate are in turn linked (via the peptide CO group) to arrays of hydrogen bonds. Three of these arrays are composed of alternating peptide units and hydrogen bonds within the solvent-exposed part of three alpha-helices and two are linked to a histidine and an arginine residue. The sulfate-binding protein bears strong similarity to the structures of four other periplasmic binding proteins solved in our laboratory which are specific for L-arabinose, D-galactose/D-glucose, leucine/isoleucine/valine and leucine. The similarity includes the ellipsoidal shape and the two globular domain structures, each domain consisting of a central beta-pleated sheet flanked by alpha-helices.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1988

Inhibitors of sterol synthesis. Concerning the structure of 15 beta-methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol, an inhibitor of cholesterol biosynthesis.

The chemical synthesis of 3 beta-p-bromobenzoyloxy-15 beta-methyl-5 alpha,14 beta-cholest-7-en-15 alpha-ol from 15 beta-methyl-5 alpha, 14 beta-cholest-7-ene-3 beta,15 alpha-diol is described. The structure of the former compound was unambiguously determined by X-ray crystallographic analysis. The space group of the crystal was P2 with unit cell parameters a = 12.611 A, b = 9.826 A, c = 13.221 A, b = 91.71 degrees and Z = 2. The structure was solved by the heavy atom method and refined to a final R of 0.041. Asymmetry parameters indicated that ring A is a symmetrical chair, that rings B and C are half chairs, and that ring D is a 15 alpha-envelope.

Anticholesteremic Agents↗
PubMed1988

Concerning the structure of 3 beta-benzoyloxy-5 beta-cholesta-8,14-diene, a major byproduct in the chemical synthesis of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one.

The X-ray crystal structure of 3 beta-(p-bromobenzoyloxy)-5 beta-cholesta-8,14-diene (space group P21, a = 10.698 A, b = 9.487 A, c = 15.024 A, beta = 96.05 degrees, Z = 2) was determined by the heavy atom method and refined to R = 0.075. This heavy atom derivative was synthesized from 5 beta-cholesta-8,14-diene-3 beta-ol, the benzoate ester of which was previously shown to be the major byproduct in the low-temperature isomerization of 7-dehydrocholesteryl benzoate in HCl/chloroform. The work presented here establishes unequivocally that the configuration of this isomerization byproduct at C-5 is 5 beta-H and that the configuration at C-17 was unchanged.

Anticholesteremic Agents↗
PubMed1988

Crystal structure of 3 beta-benzoyloxy-6 alpha-chloro-5 alpha-cholest-7-ene, a key intermediate in the chemical synthesis of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one.

The X-ray crystal structure of 3 beta-benzoyloxy-6 alpha-chloro-5 alpha-cholest-7-ene (IV) was determined by the heavy atom method and refined to R = 0.063 (space group P21, a = 11.364, b = 11.089, c = 12.232, beta = 99.43 degrees, Z = 2). IV was previously shown to be an important intermediate in the acid-catalyzed isomerization of 7-dehydrocholesteryl benzoate. The present work unequivocally establishes the location of the double bond and the configuration of the chlorine of IV, information which is essential to the correct formulation of the mechanism of this reaction.

Anticholesteremic Agents↗
PubMed1987

15 beta-Methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol. Synthesis, structure, and inhibition of sterol synthesis in animal cells.

Treatment of 3 beta-benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene with methyl magnesium iodide gave, as the major product, 15 beta-methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol. The product was characterized as the free sterol and in the form of its 3 beta-acetoxy and 3 beta-p-bromobenzoate derivatives. Unambiguous assignment of structure was based upon X-ray analysis of the latter derivative. 15 beta-Methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol was found to be a potent inhibitor of sterol synthesis in cultured mammalian cells. The 15 beta-methyl-3 beta,15 alpha-dihydroxysterol caused a 50% reduction of the level of HMG-CoA reductase activity and a 50% reduction in the incorporation of labeled acetate into digitonin-precipitable sterols in L cells at a concentration of 3.0 x 10(-6) M.

Animals↗
PubMed1986

Crystallization and x-ray diffraction studies of a phosphate-binding protein involved in active transport in Escherichia coli.

We have obtained single crystals of a phosphate-binding protein (Mr = 34,400) that serves as initial receptor in osmotic shock-sensitive active transport in Escherichia coli. The crystals, suitable for high resolution crystallographic analysis, belong to the space group P2(1)2(1)2(1). The unit cell has dimensions of a = 41.97, b = 64.66, and c = 124.6 A and contains four protein molecules. Including this phosphate-binding protein, there are now a total of six different binding protein structures currently under investigation in our laboratory, the others being those specific for L-arabinose, D-galactose, D-maltose, sulfate, or leucine/isoleucine/valine.

Carrier Proteins↗
PubMed1983

Rates of ligand binding to periplasmic proteins involved in bacterial transport and chemotaxis.

The ligand reactions of three binding proteins involved in bacterial transport and chemotaxis have been examined by stopped flow, rapid mixing techniques. The processes measured were: L-arabinose, D-galactose, and D-fucose binding to the Escherichia coli L-arabinose-binding protein; L-histidine binding to the Salmonella typhimurium L-histidine-binding protein; and D-maltose, maltotriose, cyclic maltohexaose, and cyclic maltoheptaose binding to the E. coli D-maltose-binding protein. Changes in tryptophan fluorescence were monitored, and the resultant time courses were analyzed quantitatively in terms of a simple one-step binding process. The fitted association rate constants for sugar binding are all about 1-3 X 10(7) M-1 s-1; variation in the affinity constants is expressed primarily by changes in the dissociation rate constants, 1-100 s-1. The sugar-binding proteins react at equal rates with the alpha and beta anomeric forms of their substrates. The ligand dissociation rates measured in vitro are consistent with the corresponding Vmax values observed for in vivo active transport. The association rate constant for the L-histidine-binding protein is 5-10 times greater than the corresponding rate constants for the sugar-binding proteins. A similar, large bimolecular rate, approximately 1 X 10(8) M-1 s-1, has been observed for the E. coli L-glutamine-binding protein (Weiner, J. H., and Heppel, L. A. (1971) J. Biol. Chem. 246, 6933-6941) and appears to reflect favorable electrostatic interactions between the charged amino acid and the surface of the protein molecule.

ATP-Binding Cassette Transporters↗
PubMed1983

Thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli.

The thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli have been studied by isothermal and scanning calorimetry. The binding reaction with arabinose is characterized by an enthalpy change of -15.3 +/- 0.5 kcal mol-1 at 25 degrees C, and a large decrease in apparent heat capacity, amounting to -0.44 +/- 0.05 kcal K-1 mol-1, which is constant over the temperature range 8 to 30 degrees C. Very similar results were obtained with D-galactose. These calorimetric results have been combined with binding constants determined by equilibrium dialysis (Clark, A. F., Gerken, T. A., and Hogg, R. W. (1982) Biochemistry 21, 2227-2233) to obtain free energy and entropy changes over the range 5 to 30 degrees C, and by extrapolation to 60 degrees C. The protein undergoes reversible unfolding on being heated with an increase in enthalpy at 53.5 degrees C of 151.8 +/- 1.1 kcal mol-1 (169.2 +/- 1.2 kcal mol-1 at 59.0 degrees C) and in apparent heat capacity of 3.16 +/- 0.07 kcal K-1 mol-1. In the presence of arabinose, the unfolding enthalpy is increased to 200.7 +/- 1.8 kcal mol-1 at 59.0 degrees C, the increase being due to the enthalpy of dissociation of the ligand which amounts to 31 kcal mol-1 at the unfolding temperature. The unfolding temperature is increased by the presence of excess arabinose or galactose, an effect which is due solely to displacement by the added ligand of the unfolding-dissociation equilibrium. The thermodynamic data are discussed in connection with the detailed structural information available for this system from x-ray crystallography (Newcomer, M. E., Gilliland, G. L. and Quiocho, F. A. (1981) J. Biol. Chem. 256, 13213-13217, and references cited therein).

Arabinose↗
PubMed1983

Leucine, isoleucine, valine-binding protein from Escherichia coli. Structure at 3.0-A resolution and location of the binding site.

The structure of the leucine, isoleucine, valine-binding protein, an integral part of the high-affinity, branched-chain aliphatic amino acid transport system in Escherichia coli, has been solved at 3.0-A resolution by x-ray crystallography. Five isomorphous heavy atom derivatives, including anomalous differences from a samarium derivative, were used. A model of the polypeptide chain backbone reveals two distinct, globular domains connected by three strands. Each domain consists of a beta-sheet core flanked by at least two helices on either side. Difference Fourier analyses of crystals soaked in L-leucine, L-isoleucine, or L-valine have located a single amino acid-binding site in the cleft formed by the two domains. Despite the lack of significant sequence homology, the bilobate and secondary structure observed were similar to that found in the structures of L-arabinose- and D-galactose-binding proteins previously determined in our laboratory.

Bacterial Proteins↗
PubMed1983

The 3 A resolution structure of a D-galactose-binding protein for transport and chemotaxis in Escherichia coli.

X-ray diffraction studies of a D-galactose-binding protein essential for transport and chemotaxis in Escherichia coli have yielded a model of the polypeptide chain backbone. An initial polyalanine backbone trace was obtained at 3.2 A resolution by the molecular replacement technique, using a polyalanine search model derived from the refined structure of the L-arabinose-binding protein. Concurrently, a 3 A resolution electron-density map of the D-galactose receptor was determined from multiple isomorphous replacement (MIR) phases. The properly transformed initial polyalanine model superimposed on the MIR electron-density map proved to be an excellent guide in obtaining a final trace. The few changes made in the polyalanine model to improve the fit to the density were confined primarily to the COOH-terminal peptide and some loops connecting the elements of the secondary structure. Despite the lack of significant sequence homology, the overall course of the polypeptide backbone of the D-galactose-binding protein is remarkably similar to that of the L-arabinose-binding protein, the first structure in a series to be solved from this family of binding proteins. Both structures are elongated (axial ratios of 2:1) and composed of two globular domains. For both proteins, the arrangements of the elements of the secondary structure in both domains are identical; both lobes contain a core of beta-pleated sheet with a pair of helices on either side of the plane of the sheet. The four major hydrophobic clusters that stabilize the structure of the L-arabinose-binding protein are also present in the D-galactose-binding protein.

Bacterial Proteins↗
PubMed1982

Hinge-bending in L-arabinose-binding protein. The "Venus's-flytrap" model.

Theoretical conformational energy calculations show that large changes in the width of the binding-site cleft in the L-arabinose-binding protein involve only modest changes in the protein internal energy. Solvation energy changes associated with such variations of the cleft width and with protein-ligand interactions are estimated to be significantly larger than the internal energy changes. These results indicate that the binding-site cleft is open in the unliganded protein and is induced to close upon ligation. This picture is consistent with experimental data on the structure and binding kinetics of the L-arabinose-binding protein and provides a physical framework for interpreting such data.

Bacterial Proteins↗