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B Richey

Publications and source records attributed to B Richey.

13 recordsLinked to original sources
PubMed1988

Interaction of an N-methylated polyamine analogue, hexamethonium(2+), with NaDNA: quantitative 14N and 23Na NMR relaxation rate studies of the cation-exchange process.

The interactions of the divalent hexamethonium (Hex2+) cation with double-helical calf thymus DNA are investigated by means of 14N NMR and, indirectly, by means of 23Na NMR. During a titration of NaDNA with HexBr2, the displacement of Na+ from DNA by Hex2+ is monitored by concurrent measurements of the Lorentzian 14N signals and the bi-Lorentzian 23Na signals. The variations in the quadrupolar relaxation rates of 14N and 23Na are analyzed according to a simple two-state model for the competition between Hex2+ and Na+ associated with DNA. From this analysis parameters characterizing the exchange process are evaluated, and the following conclusions are drawn: (1) The association of one Hex2+ displaces 1.7-2.0 sodium ions from the vicinity of the DNA. (2) Cation accumulation near DNA neutralizes approximately half of the phosphate charge at all points in the titration. (3) The exchange coefficient characterizing the displacement of Na+ by Hex2+ is of the same order of magnitude as the exchange coefficients determined by NMR for other divalent cations such as Mg2+ and putrescine. These findings imply that the interaction of Hex2+ with DNA is primarily electrostatic in character. The transverse and longitudinal relaxation rates observed for 14N are analyzed under the assumption that the quadrupolar relaxation processes of 14N in Hex2+ associated with DNA can be characterized by a single-exponential correlation function with correlation time tau NB. The resulting value of tau NB, 7.8 +/- 0.8 ns, is 3 orders of magnitude greater than that estimated for Hex2+ in the absence of DNA and is only 3-4 times greater than correlation times reported for 23Na and other quadrupolar cations near DNA. These comparisons indicate that the observed enhancements in the relaxation rates of 14N are due mainly to slowing of the motions that modulate its quadrupolar interactions in Hex2+ near DNA. The magnitudes of tau NB and of the quadrupolar coupling constant of Hex2+ associated with DNA are consistent with the conclusion that this association is primarily electrostatic.

Animals↗
PubMed1987

Variability of the intracellular ionic environment of Escherichia coli. Differences between in vitro and in vivo effects of ion concentrations on protein-DNA interactions and gene expression.

Effects of changes in intracellular ion concentrations on the interactions of Escherichia coli lac repressor with lac operator mutants and on the interactions of RNA polymerase with various promoters have been investigated in vivo. The intracellular ionic environment was reproducibly varied by changing the osmolality of the 4-morpholinepropanesulfonic acid minimal growth medium. As the osmolality of the growth medium is varied from 0.1 to 1.1 osmolal, the total intracellular concentration of K+ increases linearly from 0.23 +/- 0.03 to 0.93 +/- 0.05 molal and the total intracellular concentration of glutamate increases linearly from 0.03 +/- 0.01 to 0.26 +/- 0.02 molal. The sum of the changes in the total concentrations of these two ions appears sufficient to compensate for a given change in external osmolality, indicating that K+ and glutamate are the primary ionic osmolytes under these conditions and that these ions are free in the cytoplasm. In support of this, in vivo 39K NMR experiments as a function of external osmolality indicate that changes in the total cytoplasmic K+ concentration correspond to changes in the free cytoplasmic K+ concentration. Extents of interaction of lac repressor and RNA polymerase with their specific DNA sites were monitored by measuring the amounts of beta-galactosidase produced under the control of these sites. For both lac repressor and RNA polymerase, it was found that formation of functional protein-DNA complexes in vivo is only weakly (if at all) dependent on intracellular ion concentration. These results contrast strongly with those obtained on these systems in vitro, which showed that both the equilibria and kinetics of binding are extremely salt-dependent. We discuss several possible mechanisms by which E. coli may compensate for the potentially disruptive effects of these large changes in the intracellular ionic environment.

Amino Acids↗
PubMed1987

Nesting: hierarchies of allosteric interactions.

A generalization of the allosteric model is presented that incorporates a hierarchy of conformational equilibria. Such a formulation draws upon structural organization already seen in many large macromolecular systems. The functional binding properties of the macromolecule reflect conformational equilibria at each structural level. Appropriate "nested" models are used to interpret structural features and functional aspects of two hemocyanin systems with a large number (12 and 24) of binding sites.

Allosteric Regulation↗
PubMed1985

Binding of oxygen and carbon monoxide to arthropod hemocyanin: an allosteric analysis.

The binding of oxygen and carbon monoxide to hemocyanin from the mangrove crab Scylla serrata and the lobster Homarus americanus has been studied by thin-layer optical absorption and front face fluorescence techniques. Three types of experiments were performed on subunit and oligomeric preparations of each hemocyanin: oxygen binding, carbon monoxide binding, and oxygen-carbon monoxide competition studies. The results obtained from the subunit preparations of dissociated oligomers from both hemocyanins show that the binding site can be ligated by either one oxygen or one carbon monoxide. The binding results obtained with the oligomeric samples of hemocyanin from both species cannot be described by the two-state MWC model [Monod, J., Wyman, J., & Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118] since the data from the three types of binding experiments cannot be fit with a single set of binding constants. The MWC model has been extended by including a third allosteric form, and an analysis based on the three-state model is able to fit the data from the three types of experiments with the same set of binding constants. The comparison of the oxygen to carbon monoxide affinity ratios (kO2/kCO) indicates that the structure around the binding site of subunits in the T form oligomer is similar to that of the free subunits. The oligomeric forms of both these hemocyanins bind carbon monoxide with a weak but definite positive cooperativity. An analysis of the affinity ratios for the T, S, and R forms suggests that the high affinity of the R form results from a specific interaction between oxygen and binding site.

Allosteric Regulation↗
PubMed1985

Generalized binding phenomena in an allosteric macromolecule.

A general macromolecular partition function is developed in terms of chemical ligand activity, temperature and pressure for systems described by an array of species which are characterized by their state of allosteric conformation and ligand stoichiometry. The effects of chemical ligand binding, enthalpy change, and volume change are treated in a parallel manner. From a broad viewpoint all of these effects can be regarded as specific cases of generalized binding phenomena. This approach provides a general method for analyzing calorimetric and ligand binding experiments. Several applications are given: (1) Thermal scanning data for tRNAphe (P.L. Privalov and V.V. Filimonov, J. Mol. Biol. 122 (1978) 447) are shown to fit a general model with six conformational states. By application of linkage theory it is shown that sodium chloride is expelled as the molecule denatures. (2) The results of calorimetric titrations on the arabinose binding protein (H. Fukada, J.M. Sturtevant and F.A. Quiocho, J. Mol. Biol. 258 (1983) 13193) are shown to fit a simple two-state allosteric model. (3) A thermal binding curve is simulated for an unusual respiratory protein, trout I hemoglobin (B.G. Barisas and S.J. Gill, Biophys. Chem. 9 (1979) 235), in order to illustrate both the similarities and differences between enthalpy and chemical ligand binding processes.

Allosteric Regulation↗
PubMed1983

Positive cooperativity in binding carbon monoxide to hemocyanin.

The binding of carbon monoxide to hemocyanin from the crab Scylla serrata has been studied by thin layer optical absorption and front face fluorescence techniques. The binding to the monomeric form is completely noncooperative whereas the binding to the native oligomeric form is found to be weakly but definitely cooperative. An analysis based on the MWC model of the oxygen and carbon monoxide binding curves indicates that the allosteric constant, L, describing the equilibrium between the 2 unligated forms is different for each ligand. This implies that at least 3 allosteric forms are needed to characterize the binding of oxygen and carbon monoxide to this hemocyanin.

Allosteric Regulation↗
PubMed1983

Thin-layer fluorescence cell for ligand binding studies.

A high precision method for measuring the binding of gaseous ligands to proteins is presented. Front face fluorescence techniques are utilized with a special thin-layer cell in order to monitor the change in fluorescence intensity caused by changing the ligand partial pressure. The method is illustrated by examining the binding of carbon monoxide to hemocyanin from the lobster Homarus americanus.

Animals↗
PubMed1982

Unusual zwitterion of D,L-beta-carboxyaspartic acid: pKa and X-ray crystallographic measurements.

An investigation of the acidic properties and molecular structure of the new natural amino acid beta-carboxyaspartic acid (Asa) is described. The four pKas of Asa were determined by using a microtitration technique and are 0.8 +/- 0.2, 2.5 +/- 0.1, 4.7 +/- 0.1, and 10.9 +/- 0.1. The three pKas of 5-hydantoinmalonic acid were similarly measured and are 1.85 +/- 0.05, 4.63 +/- 0.05, and 10.20 +/- 0.05. 5-Hydantoinmalonic acid was used as a model for Asa with peptide bonds. Asa crystallizes in the monoclinic space group Cc with four molecules per unit cell of dimensions a = 13.112 (3) A, b = 8.207 (3) A, and c = 7.292 (2) A and beta = 108.03 (2) degrees. The structure was solved by direct methods and refined to final values for the discrepancy indices of R = 0.029 and wR = 0.036. The two molecules of Asa are linked by a very strong hydrogen bond between one of the beta-carboxyls and the alpha-carboxyl group of an adjacent molecule. Analysis of the pKa data indicates that the predominate zwitterion in solution results from ionization of a beta-carboxyl group. The X-ray data indicate that in the solid state the negative charge of the zwitterion is distributed approximately equally between one of the beta-carboxyls and the alpha-carboxyl group.

Aspartic Acid↗
PubMedDate not supplied

Triple-point behavior of human haemoglobin.

The recent crystallographic observation of Brzozowski et al. demonstrates the existence of half-oxygenated haemoglobin crystals. At first sight this seems to imply the discovery of a unique molecule, defined by exactly two bound oxygens. However, the formation of crystals with a specific degree of ligation is predicted to occur when two distinct crystalline phases coexist, namely at the triple point. It seems likely that the crystals studied by Brzozowski et al. were selected from a mixture of T and R crystalline forms obtained in conditions where the triple point existed. In general, the equilibria between haemoglobin solid and liquid phases are governed by the chemical potential of a control ligand and the ligand-binding properties of each phase. We report here that a unique situation arises when there are two different crystalline phases, each with characteristic oxygen-binding properties. One can then predict the existence of a triple point, defined by a specific oxygen partial pressure, where both solid phases coexist. At this point the degree of oxygen saturation in each solid phase is uniquely specified. These considerations could explain the specifically ligated crystals found by Brzozowski et al.

Crystallization↗