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At least 793 records · Page 44Linked to original sources
PubMed1985

Stoichiometric and electrostatic characterization of calcium binding to native and lipid-substituted adenosinetriphosphatase of sarcoplasmic reticulum.

The stoichiometry of calcium binding to specific sites (i.e., those producing enzyme activation) was found to be 8-10 nmol/mg protein in native sarcoplasmic reticulum vesicles, and 13.9-15.4 nmol/mg of ATPase purified by non-ionic detergent solubilization and anion exchange chromatography. Parallel measurements of phosphoenzyme yielded levels of 4.0-4.9 and 6.0-7.7 nmol/mg of protein in the two preparations, respectively, demonstrating that each 115 kDa ATPase chain includes one catalytic site and two calcium binding sites. The apparent association constant, K = (6 +/- 2) X 10(5) M-1, and the binding cooperativity, nH = 1.9, were unchanged when measurements were carried out with native sarcoplasmic reticulum vesicles and when the membrane surface charge was altered by lipid substitution with phosphatidylcholine or phosphatidylserine, at neutral pH in the presence of 10 mM MgCl2 and 80 mM KCl. On the other hand, the apparent association constant was increased in the absence of Mg2+ or, to a lesser extent, in the absence of monovalent cations. It was also observed that the cooperative character of the calcium binding isotherms was reduced in low ionic-strength media. Analysis of the electrostatic effects indicates that the calcium-binding domain is shielded from the membrane phospholipid surface charge by virtue of its location within the ATPase protein. The effects of various electrolytes are attributed to monovalent-cation binding in the calcium-binding domain. The apparent loss of cooperativity of the calcium binding isotherms at low ionic strength is attributed to a progressive displacement of the titration curve which is minimal at low degrees of saturation and becomes larger at higher degrees of saturation. This behavior is described quantitatively by the progressive effect of calcium binding on an electrostatic potential generated by localized protein charge densities within, or near, the calcium-binding domain.

Adenosine Triphosphatases↗
PubMed1991

Erythrocyte aggregation: bridging by macromolecules and electrostatic repulsion by sialic acid.

Relation between aggregating force (of fibrinogen and IgG) and disaggregating force (due to electrostatic repulsion among erythrocytes) in erythrocyte aggregation was investigated with a rheoscope combining a video camera, an image analyzer and a computer. (i) Erythrocyte aggregation was augmented with the increase of molecular weight of bridging macromolecules as far as examined for fibrinogen and the degradation products and IgG and the related macromolecules, and the augmentation seemed to be dependent on the molecular length of macromolecules. In accelerating the erythrocyte aggregation, fibrinogen was more effective than IgG, and some interaction between fibrinogen and IgG in their coexistence was suggested. (ii) The decrease of sialic acid content on the erythrocyte surface accelerated IgG-induced erythrocyte aggregation much greater than fibrinogen-induced one. (iii) Counteraction between aggregating force and disaggregating force in leading to erythrocyte aggregation was discussed relating to molecular length of bridging macromolecule and electrostatic repulsive force by sialic acid.

Adult↗
PubMed1991

The role of electrostatic forces in the interaction between the membrane and cytoskeleton of human erythrocytes.

Evidence is presented that electrostatic forces play a major role in the interaction between the cell membrane and cytoskeleton of human erythrocytes. Experiments were carried out on the effects of ionic strength variation, Ca2+ and Mg2+ ions, dimethonium ion and lipophilic ions on the release of spectrin from the erythrocyte ghost. In addition it was shown that the release of spectrin for fixed Ca2+ or Mg2+ concentration shows a maximum as a function of Na+ concentration. All results are consistent with the existence of a repulsive electrostatic force between membrane and cytoskeleton.

Aspirin↗
PubMed1991

A microscopic electrostatic model for the amphotericin B channel.

A microscopic model of an amphotericin B channel is proposed. The structure of the pores is generated using the atomic coordinates of the molecule in the structure determined experimentally by X-ray diffraction. The net charges of the atoms are determined by Mulliken analysis. With these charges the electrostatic energy profiles are calculated for a monovalent ion passing through the channels formed by different number of antibiotic molecules having different radii. The water inside the channel was considered through a continuum medium using the dielectric constant of the bulk, and the membrane contribution was included using the virtual images of the pore in a dielectric slab of epsilon = 3. The model satisfactorily explains the permeability and selectivity characteristics as well as other observations yet unexplained. The electrostatic profiles obtained reinforce the hypothesis of the existence of channels formed by a variable number of units.

Amphotericin B↗
PubMed1995

Active transport of ions across membranes: energetic role of electrostatics and binding site asymmetry.

The active transport of ions across a membrane by an ATP-driven electrogenic ion pump is often described by an 'alternate access' model. The position of the binding site is assumed to be unchanged as the binding cavity opens alternatively to the uptake and discharge sides of the membrane. The ion binding affinity is higher on the uptake side of the membrane than on the discharge side. This difference in affinities is related to the maximum transport rate and to the efficiency with which ATP hydrolysis is coupled to active transport. Here we examine the electrostatic contribution to binding affinities, using a simple geometry for a model membrane-protein system, a continuum dielectric approximation, and a numerical method to calculate binding energy as a function of the binding site location. If the binding site is located asymmetrically, being further from the uptake side of the membrane than from the discharge side, there is a significant difference in binding free energy between the uptake and discharge states. This asymmetry can produce differences in affinities that are consistent with those measured for biological active transport systems. These results may account for the observed asymmetric location of the calcium binding site in the calcium ATPases from sarcoplasmic reticulum and from the plasma membrane. Electrostatic energy differences associated with binding site asymmetry may be a general feature of electrogenic transmembrane ion pumps.

Adenosine Triphosphatases↗
PubMed1978

The acceleration of methanesulfonylation of acetylcholinesterase with cationic accelerators as an electrostatic effect.

1. In order to check our hypothesis of the electrostatic nature of the acceleration of methanesulfonylation of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) with cationic accelerators, equations were solved for methane-sulfonylation with two accelerators and the reaction was studied in the presence of some single accelerators, including the sodium cation, and in the presence of two acclerators simultaneously. 2. The second-order rate constants for methanesulfonylation of the complexes between the enzyme and accelerators decamethonium, tetraethylammonium and tetramethylammonium are 90, 88 and 17 1 - mol-1 - s-1, respectively, which corresponds to a maximal acceleration of 29, 28 and 5.5 times, respectively. The dissociation constants for the binding of these accelerators to the enzyme, obtained from our acceleration experiments, are 3.7 - 10(-6), 3.2 - 10(-4) and 1.4 - 10(-3) M, respectively. These values are in good agreement with the dissociation constants of these ligands as inhibitors of acetylcholinesterase. It is interesting to note that the sodium cation also accelerates the methane-sulfonylation up to around three times, the corresponding second-order rate constant and the dissociation constant being 10 1 - mol-1 - s-1 and 1.3 M, respectively. 3. All tested cations compete in the acceleration with each other; they seem to accelerate the reaction in the same way and from the same site, the catalytic anionic site. 4. These findings confirm the hypothesis of the electrostatic nature of acceleration.

Acetylcholinesterase↗
PubMed1980

Catalytic properties and electrostatic potential of charged immobilized enzyme derivatives. Pyruvate decarboxylase attached to cationic polystyrene beads of different charge densities.

Pyruvate decarboxylase has been covalently attached to positively charged macroporous polystyrene resins. The changes in the functional behaviour of the enzyme resulting from the electrostatic interaction of the cationic carrier polymer and the anionic substrate have been studied as function of the fixed charge concentration of the carrier. The findings confirm the connection of two widely used theoretical expressions relating electrostatic parameters with Michaelis constants.

Carboxy-Lyases↗
PubMed1988

Formation of electrostatically-stabilized complex at low ionic strength inhibits interprotein electron transfer between yeast cytochrome c and cytochrome c peroxidase.

Electron transfer from yeast ferrous cytochrome c to H2O2-oxidized yeast cytochrome c peroxidase has been studied using flash photoreduction methods. At low ionic strength (mu less than 10 mM), where a strong complex is formed between cytochrome c and peroxidase, electron transfer occurs rather slowly (k approximately 200s-1). However, at high ionic strength where the electrostatic complex is largely dissociated, the observed first-order rate constant for peroxidase reduction increases significantly reaching a concentration independent limit of k approximately 1500 s-1. Thus, at least in some cases, formation of an electrostatically-stabilized complex can actually impede electron transfer between proteins.

Cytochrome c Group↗
PubMed1984

Potential-derived point-charge model study of electrostatic interactions in DNA base components.

Ab initio electrostatic potentials obtained using STO-3G wavefunctions for guanine, cytosine, adenine, and thymine are used to calculate potential-derived (PD) point charges for these base components. Calculated PD point charges are used to estimate the electrostatic contributions to hydrogen-bonding and stacking interaction energies of ten sequence isomers of B-DNA. These estimates are in excellent agreement with the results of the more elaborate segmental multipole moment expansion technique.

Adenine↗
PubMed1989

Substitution of arginine for lysine 134 alters electrostatic parameters of the active site in shark Cu,Zn superoxide dismutase.

The complete amino acid sequence was determined for the Cu,Zn superoxide dismutase from the shark Prionace glauca. The active site region shows the substitution of an Arg for Lys at position 134, which is important for electrostatic facilitation of the diffusion of O2- to the catalytically active copper. This change may be related to observed alterations of electrostatic parameters of the enzyme (pK of the pH dependence of the enzyme activity, rate of inactivation by H2O2), although it preserves a high efficiency of dismutation at neutral pH.

Amino Acid Sequence↗
PubMed1993

Combined effects of diffusional hindrances, electrostatic repulsion and product inhibition on the kinetic properties of a bound acid phosphatase.

The kinetic properties of a soybean cell wall phosphatase were studied and compared under different environmental conditions. The native enzyme isolated from the wall exhibits classical Michaelis-Menten kinetics. When buried into the cell wall, its natural environment, the enzyme mimics an apparent negative cooperativity. This deviation from hyperbolic dependence of the activity vs. the substrate concentration may result from an heterogeneous distribution of the enzyme molecules between the surface and the inside of the wall, where an electrostatic partition effect takes place. Cell wall fragments compacted on a Millipore-type membrane allow the study of the dual effects of diffusional hindrances and electrostatic interactions on the global kinetic behavior. By increasing the ionic strength, partition effects can be suppressed and therefore diffusional effects alone can be taken into account. On the one hand, diffusion and partition act individually and synergetically to decrease the apparent global affinity of phosphatase with respect to glucose 6-phosphate. On the other hand, product inhibition by inorganic phosphate is subjected to dual effects from diffusion through accumulation and partition through repulsion. A simple diffusion-partition reaction model accounts qualitatively and quantitatively for the experimental observations.

Acid Phosphatase↗
PubMed1996

Lateral interaction between electrostatically adsorbed and covalently immobilized proteins on the surface of cation-exchange sorbents.

This paper examines the nature of chromatographic separations on a weak cation-exchange material in which immobilized proteins coats 50% or less of the sorbent surface. It was found that although these sorbents still function as cation exchangers, covalently immobilized proteins frequently contribute to the ion-exchange behavior of some protein analytes. Chromatographic retention of analytes was equal to or greater on immobilized protein derivatized columns than underivatized sorbents. Anionic proteins, in contrast, were not adsorbed, indicating that immobilized proteins were acting synergistically with ionic stationary phase groups to enhance retention. It was concluded that electrostatic adsorption is a prerequisite for analyte protein/immobilized protein interactions of sufficient magnitude to impact ion-exchange separations. Large differences in protein resolution were observed on columns that were identical in all respects except for the immobilized protein, further confirming that analyte/immobilized protein interactions were unique to the interacting pair. The extent of interaction was also influenced by concentration of the immobilized protein in the case of lysozyme. Interactions between the analyte and immobilized protein were found to occur between both the same two proteins and dissimilar species. It was concluded that these phenomena are due to lateral interactions between immobilized proteins and analyte proteins subsequent to electrostatic adsorption of the analyte on the underivatized surface of ion-exchange sorbents.

Acrylic Resins↗
PubMed1985

Electrostatic field of the large fragment of Escherichia coli DNA polymerase I.

The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy.

DNA Polymerase I↗
PubMed1991

Role of electrostatic repulsion in the acidic molten globule of cytochrome c.

The molten globule has been assumed to be a major intermediate state of protein folding. To extend our understanding of protein folding it is important to elucidate the thermodynamic mechanism of conformational stability of the molten globule. To clarify the role of electrostatic charge repulsion in the stability of the acidic molten globule state, we prepared a series of acetylated horse ferricytochrome c species with various degrees of charge repulsion. On the basis of circular dichroism measurement, we show that the stability of the acidic molten globule is determined by a balance of electrostatic repulsions between positive residues, which favor the extended conformation, and the opposing forces, which stabilize the molten globule. These results provide a clear example of charge repulsions producing unfolding of the compact protein structure, and suggest that the reversibly denatured conformation of ferricytochrome c under physiological conditions (i.e. neutral pH, ambient temperature and no denaturant) is the molten globule.

Acetylation↗
PubMed1991

Mapping electrostatic interactions in macromolecular associations.

In the association of electron transfer proteins, electrostatics has been proposed to play a role in maintaining the stability and specificity of the biomolecular complexes formed. An excellent model system is the interaction between mammalian cytochrome b5 and cytochrome c, in which the X-ray structures of the individual components reveal a complementary asymmetry of charges surrounding their respective redox centers. Determining the exact extent of the electrostatic interactions and identifying the specific residues involved in the formation of the electron transfer complex has proved more elusive. We report herein the utilization of high-pressure techniques, together with site-directed mutagenesis, to provide a map of the interaction domains in biomolecular complex formation. The application of high pressure disrupts macromolecular associations since dissociation of the complex results in a decreased volume of the system due to the solvation of charges that had been previously sequestered in the interface region and force solvation of hydrophobic surfaces. Site-directed mutagenesis of a totally synthetic gene for rat liver cytochrome b5, which expresses this mammalian protein in Escherichia coli as a hemecontaining soluble component, was used to selectively alter negatively charged residues of cytochrome b5 to neutral amide side-chains. We have demonstrated that the interaction domain of cytochrome b5 with cytochrome c can be mapped from a comparison of dissociation volumes of these modified cytochrome b5-cytochrome c complexes with the native complex. Using these techniques we can specifically investigate the role of particular residues in the equilibrium association of these two electron transfer proteins. Single-point mutations in the interaction domain give nearly identical effects on the measured dissociation volumes, yet removal of acidic residues outside the recognition surface yield volumes similar to wild-type protein. Multiple mutations in the proposed protein-protein interaction site are found to allow greater solvent-accessibility of the interface as reflected in a diminution in the volume changes on subsequent charge removal. This is indicative that the interprotein salt-bridges in this complex provide a mechanism for a greater exclusion of solvent from the interfacial domain of the complex, resulting in a more stable association.

Amino Acids↗
PubMed1992

Model for the differential stabilities of rhinovirus and poliovirus to mild acidic pH, based on electrostatics calculations.

Previous calculations of electrostatic interactions in the rhinovirus capsid have identified a subset of histidine residues, paired with lysine or arginine, that may be involved in pH-induced conformational changes related to viral uncoating. Further calculations with the finite difference method, accounting for the dielectric environment of the ionizable groups, suggest that charge burial in the crystal conformation will prevent protonation of these histidine residues in the pentamer-pentamer interface. Calculations with a modelled pentamer-pentamer interface in which three beta-strands are removed recover mildly acidic pKa values for the histidines. These results are discussed in the context of the structural interactions of these three beta-strands, which form a beta-sheet extension from the rest of the capsid, and with regard to the conformation of the homologous beta-sheet extension in poliovirus, which also possesses homologous histidine-lysine/arginine pairs. A model is developed in which the structural stability of the beta-sheet extension is related to the difference in acid stability of rhinovirus and poliovirus. It is suggested that, for poliovirus prior to cell receptor binding, the beta-sheet extension is stable at pH 3, the pentamer-pentamer interface histidines remain buried, and the virus is acid-stable. Cell receptor binding of poliovirus destabilizes the beta-sheet extension and the acid lability that is proposed to result could be involved in viral uncoating. For rhinovirus it is suggested that the observed conformational change in the absence of cell receptor binding involves a further acidic pH-activated process or conformational fluctuations that rearrange the beta-sheet extension and expose the pentamer-pentamer interface histidine residues to the acidic medium. Sequence analysis and electrostatics calculations reveal an aspartic acid in the beta-sheet extension that may have different pKa values in rhinovirus and poliovirus.

Amino Acid Sequence↗
PubMed1992

Electrostatic calculations of the pKa values of ionizable groups in bacteriorhodopsin.

The effects of solvation and charge-charge interactions on the pKa of ionizable groups in bacteriorhodopsin have been studied using a macroscopic dielectric model with atom-level detail. The calculations are based on the atomic model for bacteriorhodopsin recently proposed by Henderson et al. Even if the structural data are not resolved at the atomic level, such calculations can indicate the quality of the model, outline some general aspects of electrostatic interactions in membrane proteins, and predict some features. The effects of structural uncertainties on the calculations have been investigated by conformational sampling. The results are in reasonable agreement with experimental measurements of several unusually large pKa shifts (e.g. the experimental findings that Asp96 and Asp115 are protonated in the ground state over a wide pH range). In general, we find that the large unfavorable desolvation energies of forming charges in the protein interior must be compensated by strong favorable charge-charge interactions, with the result that the titrations of many ionizable groups are strongly coupled to each other. We find several instances of complex titration behavior due to strong electrostatic interactions between titrating sites, and suggest that such behavior may be common in proton transfer systems. We also propose that they can help to resolve structural ambiguities in the currently available density map. In particular, we find better agreement between theory and experiment when a structural ambiguity in the position of the Arg82 side-chain is resolved in favor of a position near the Schiff base.

Bacteriorhodopsins↗
PubMed1983

The influence of hydrophobic ions and dipolar molecules on the electrostatic barrier in biomembranes.

To calculate the electric field inside a membrane the aqueous phase can be approximated by a conductor since the dielectric constant of water is much larger than that of the membrane. Then, using the method of image charges, ions adsorbed inside the membrane can be considered as dipoles and dipolar molecules adsorbed inside the membrane may similarly be regarded as sets of two similarly oriented dipoles. The microscopic interactions and, therefore, the spatial correlations of the adsorbed species can then be obtained. Together with the Gouy theory for the diffuse double layer these results allow the determination of the adsorbed phase--aqueous phase equilibrium. From the densities and spatial correlations of the adsorbed ions and dipolar species, their influence upon the electrostatic barrier as experienced by an ion translocating the membrane can be calculated. Changes observed in the relaxation time and initial conductance of translocating hydrophobic ions in voltage-pulse experiments on bilayer membranes are predicted using this model of the electrostatic barrier. In addition, an equation giving the surface tension as a function of the (non-ideal) adsorption of hydrophobic ions and dipoles is derived.

Adsorption↗