Theoretical research on structures of gamma-aminobutyric acid and glutamic acid in aqueous conditions

Even though glutamic acid contains only one more carboxyl group than gamma-aminobutyric acid (GABA), these neurotransmitters are recognized by their own specific receptors. To understand the ligand-recognition mechanism of the receptors, we must determine the geometric and electronic structures of GABA and glutamic acid in aqueous conditions using the ab initio calculation. The results of the present study showed that the stable structure of GABA was the extended form, and it attracted both cations and anions. Glutamic acid only attracted cations and was stabilized in four forms in aqueous conditions: Type 1 (an extended form), Type 2 (a rounded form), and Types 3 and 4 (twisted forms of Type 1). The former two types had low energy and the energy barrier between them was estimated to be small. These results showed that most free glutamic acid is present as Type 1, Type 2, and transient forms. The present results therefore suggest that the flexibility of the geometric structures of ligands should be taken into account when we attempt to elucidate the mechanism of recognition between ligands and receptors, in addition to the physicochemical characteristics of ligands and receptors.     

Spatial Separation of Molecular Conformers and Clusters

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.     

Relevance of charged groups for the integrity of the S-layer from Bacillus coagulans E38-66 and for molecular interactions.

In this paper, the importance of charged amino and carboxyl groups for the integrity of the cell surface layer (S-layer) lattice from Bacillus coagulans E38-66 and for the self-assembly of the isolated subunits was investigated. Amidination of the free amino groups which preserved their positive net charge had no influence on both. On the other hand, acetylation and succinylation, which converted the amino groups into either neutral or negatively charged groups, and amidation of carboxyl groups were accompanied by the disintegration or at least by the loss of the regular structure of the S-layer lattice. Treatment of S-layer monolayers with the zero-length cross-linker carbodiimide led to the introduction of peptide bonds between activated carboxyl groups and amino groups from adjacent subunits. This clearly indicated that in the native S-layer lattice the charged groups are located closely enough for direct electrostatic interactions. Under disrupting conditions in which the S-layer polypeptide chains were unfolded, 58% of the Asx and Glx residues could be amidated, indicating that they occur in the free carboxylic acid form. As derived from chemical modification of monolayer self-assembly products, about 90% of the lysine and 70% of the aspartic and glutamic acid residues are aligned on the surface of the S-layer protein domains. This corresponded to 45 amino groups and to 63 carboxyl groups per S-layer subunit. Labelling experiments with macromolecules with different sizes and charges and adsorption studies with ion-exchange particles revealed a surplus of free carboxyl groups on the inner and on the outer faces of the S-layer lattice. Since the carboxyl groups on the outer S-layer face were accessible only for protein molecules significantly smaller then the S-layer protomers or for positively charged, thin polymer chains extending from the surface of ion-exchange beads, the negatively charged sites must be located within indentations of the corrugated S-layer protein network. This was in contrast to the carboxyl groups on the inner S-layer face, which were found to be exposed on elevations of the S-layer protein domains (D. Pum, M. Sára, and U.B. Sleytr, J. Bacteriol. 171:5296-5303, 1989).     

Structural transformation of apocytochrome c induced by alternating copolymers of maleic acid and alkene

Apocytochrome c interacts with two copolymers: poly(isobutylene-alt-maleic acid) (PIMA) and poly(1-tetradecene-alt-maleic acid) (PTMA). The interaction leads to apocytochrome c, a conformational change from random coil to alpha-helical structure. The alpha-helix content is influenced by the copolymer concentration, the length of alkyl chain of the copolymers, and pH of the medium. The electrostatic attraction between the copolymer and protein is an indispensable factor for the folding of the protein at acid pH. The hydrophobic interaction is an important factor over the entire pH range, especially when both the copolymer and protein carry negative charges at alkaline pH. The electrostatic and hydrophobic attractions between the copolymer and protein exclude water molecules, promoting the formation of hydrogen bonds within the helical structure. On the other hand, the hydrogen bonds formed between the ionized carboxyl of the copolymer and the amide of the protein partly restrain the formation of hydrogen bonds within the helical structure when the copolymer concentration is higher at pH 6.5 and 10.5.     

Redistribution of the electric field within the pore contributes to the voltage-dependence of mitochondrial porin channel.

The effects of pH on the integral conductance and on the properties of single channels induced by porin from rat liver mitochondria in a lipid bilayer have been studied. When the membrane potential increases, the conductance of the multi-channel membrane decreases more sharply at acidic pH than at neutral or basic pH. The channel is shown to have several states with different conductance and selectivity. The number of levels and their conductance do not depend on pH, while the selectivity as well as the dependence of steady-state probabilities of different levels on the membrane potential are substantially affected by a pH change. This dependence curve steepens in the pH region where charges of carboxyl groups of aspartic and glutamic amino acids are neutralized. It is concluded that at neutral pH the channel gate is controlled by a great number of the positively and negatively charged groups. The high steepness of the conductance-voltage curve in the acidic region suggests that at least 60 positive charges participate in controlling the channel gate. This number, compared with that of the positively charged side chain amino acids per channel, according to the amino acid analysis of the porin, led us to conclude that almost all amino groups of the channel former must pass through the entire membrane potential difference upon random motion of the channel among the states. The assumption that channel closing leads to redistribution of the electric field within the pore, changing the energy of the charges on the voltage sensor, may be the only explanation of this phenomenon.     

Use of reversed-phase high-performance liquid chromatography for simultaneous determination of glutamine synthetase and glutamic acid decarboxylase in crude extracts

Glutamine and γ-aminobutyric acid (GABA), formed from glutamic acid in crude tissue extracts by glutamine synthetase and glutamic acid decarboxylase respectively, were separated by derivatization with dansyl chloride followed by reversed-phase high-performance liquid chromatography on an Altex Ultrasphere ODS-5 column. The mobile phase was a gradient of 100 mM potassium dihydrogen phosphate (pH 2.1) with 0–40% acetonitrile. The amounts of glutamine and GABA formed from glutamic acid were determined under different reaction conditions.     

Inhibitory effect of carboxylic acid group on hERG binding

Drug-induced QT prolongation arising from drugs' blocking of hERG channel activity presents significant challenges in drug development. Many, but not all, of our benzamidine-containing factor Xa inhibitors were found to have high hERG binding propensity. However, incorporation of a carboxylic acid group into these benzamidine molecules generally leads to hERG inactive compounds regardless where the carboxyl group is tethered within the molecules. The inhibitory effect of a carboxylic acid group on hERG binding has also been observed in many series of diverse structural scaffolds (including non-amidines). These findings suggest that the negatively charged carboxylate group causes unfavorable interaction within hERG channel binding cavity by electrostatic interaction.     

The influence of electrostatic interactions on partition in aqueous polyethylene glycol/dextran biphasic systems: Part II

In aqueous polyethylene glycol/dextran two-phase systems, the hydrophobicity, free volume, surface tension, and interfacial tension of the phases in equilibrium were measured as a function of pH and ionic strength. These parameters were found to change with pH, but the pattern and magnitude cannot explain the unusual partition of charged macromolecules, observed previously. The electrostatic potential difference was determined by a new experimental approach based on the measurement of the pH difference between the phases at equilibrium. In polyethylene glycol/dextran systems containing sodium chloride as ionized species, the electrostatic potential is not constant in the pH range 2 to 11. The partition behavior of charged macromolecules and its dependence on pH can be explained by the combined action of charge and phase potential. This conclusion was tested with poly-L-glutamate, which partitioned as predicted and in a pattern opposite to positively charged macro- molecules. (c) 1995 John Wiley & Sons, Inc.     

Protein engineering of cytochrome c by semisynthesis: substitutions at glutamic acid 66

We have used protein semisynthesis to prepare four analogues of horse cytochrome c, in which the glutamic acid residue at position 66 has been removed and replaced by norvaline, glutamine, lysine and, as a methodological control, glutamic acid. This residue is quite strongly conserved in mitochondrial cytochrome c, and forms part of a cluster of acidic residues that occurs in all cytochromes c but whose function is obscure. Comparative studies of the physical and biochemical properties of the analogues have now disclosed two specific roles for Glu66 in the protein. It contributes significantly to the stabilization of the active conformation of the protein, probably by salt bridge formation, and it appears to participate in the redox-state-dependent ATP-binding site of cytochrome c. Our results also support two general views of the role of surface charged residues in cytochrome c, namely that their disposition influences both redox potential, through the electrostatic field felt at the redox centre, and the kinetics of electron transfer, through the dipole moment they generate.     

Direct identification of lysine-33 as the principal cationic center of the omega-amino acid binding site of the recombinant kringle 2 domain of tissue-type plasminogen activator.

We have generated site-specific mutants of the kringle 2 domain of tissue-type plasminogen activator [( K2tPA]) in order to identify directly the cationic center of the protein that is responsible for its interaction with the carboxyl group of important omega-amino acid effector molecules, such as epsilon-amino caproic acid (EACA). Molecular modeling of [K2tPA], docked with EACA, based on crystal structures of the kringle 2 region of prothrombin and the kringle 4 domain of human plasminogen, clearly shows that Lys33 is the only positively charged amino acid in [K2tPA] that is sufficiently proximal to the carboxyl group of the ligand to stabilize this interaction. In order to examine directly the importance of this particular amino acid residue in this interaction, we have constructed, expressed, and purified three recombinant (r) mutants of [K2tPA], viz., Lys33Thr, Lys33Leu, and Lys33Arg, and found that only the last variant retained significant ability to interact with EACA and several of its structural analogues at neutral pH. In addition, another mutated r-[K2tPA], i.e., Lys33His, interacts very weakly with omega-amino acids at neutral pH and much more strongly at lower pH values where His33 would be expected to undergo protonation. This demonstrates that any positively charged amino acid at position 33 satisfies the requirement for mediation of significant bindings to this class of molecules. Since, in other kringles, positively charged residues at amino acid sequence positions homologous to Lys68, Arg70, and Arg71 of [K2tPA] have been found to participate in kringle interactions with EACA-like compounds, we have also examined the binding of EACA, and some of its analogues, to three additional r-[K2tPA] variants, i.e., Lys68Ala, Arg70Ala, and Arg71Ala. In each case, binding of these omega-amino acids to the variant kringles was observed, with only the Lys68Ala variant showing a slightly diminished capacity for this interaction. These investigations provide clear and direct evidence that Lys33 is the principal cationic site in wild-type r-[K2tPA] that directly interacts with the carboxyl group of omega-amino acid effector molecules.     

Electric dipole moments of the fluorescent probes Prodan and Laurdan: experimental and theoretical evaluations

Several experimental and theoretical approaches can be used for a comprehensive understanding of solvent effects on the electronic structure of solutes. In this review, we revisit the influence of solvents on the electronic structure of the fluorescent probes Prodan and Laurdan, focusing on their electric dipole moments. These biologically used probes were synthesized to be sensitive to the environment polarity. However, their solvent-dependent electronic structures are still a matter of discussion in the literature. The absorption and emission spectra of Prodan and Laurdan in different solvents indicate that the two probes have very similar electronic structures in both the ground and excited states. Theoretical calculations confirm that their electronic ground states are very much alike. In this review, we discuss the electric dipole moments of the ground and excited states calculated using the widely applied Lippert–Mataga equation, using both spherical and spheroid prolate cavities for the solute. The dimensions of the cavity were found to be crucial for the calculated dipole moments. These values are compared to those obtained by quantum mechanics calculations, considering Prodan in vacuum, in a polarizable continuum solvent, and using a hybrid quantum mechanics–molecular mechanics methodology. Based on the theoretical approaches it is evident that the Prodan dipole moment can change even in the absence of solute–solvent-specific interactions, which is not taken into consideration with the experimental Lippert–Mataga method. Moreover, in water, for electric dipole moment calculations, it is fundamental to consider hydrogen-bonded molecules.     

Hormone mediated changes of brain glutamic acid system in amino acid imbalance

The effects of adrenal cortical hormone and thyroxine on brain glutamic acid, gamma-amino butyric acid (GABA) and glutamine were studied in rats fed on the amino acid imbalanced diet (8% casein diet supplemented with 0.3% L-threonine). The studies revealed that the decrease in brain glutamic acid and GABA levels in threonine imbalance was recovered by hydrocortisone supplementation. The increased level of brain glutamine in threonine imbalance could not, however, be reversed by hydrocortisone supplementation. Thyroxine supplementation was found to have no impact on any of the members of glutamic acid family in the brain of rats receiving the threonine-imbalanced diet. It was suggested that the decreased levels of brain glutamic acid and GABA in threonine imbalance were caused by diminished adrenal cortical function and the influence of adrenal cortical hormone could be suggested to reside at the level of formation of both glutamic acid and GABA.     

Inhibition of crayfish glutamic acid decarboxylase by structural analogs of the substrate and product

Crayfish glutamic acid decarboxylase (GAD) is inhibited by some aliphatic carboxylic acid analogs of glutamate and gamma-amino-n-butyric acid (GABA). Variations in the length of the carbon skeleton, substitution of a keto for a methylene group, replacement of the carboxyl group or attachment of a bulky basic moiety to the amino terminus of GABA all lead to a drastic reduction in its inhibitory activity. Substitution of a methyl group for the amino group of GABA is a permissible alteration which does not reduce the inhibitory potency. Some structural analogs of glutamate are inhibitory also, particularly if they possess a comparable carbon skeleton and a keto group in the alpha position or a sulfhydryl group. Most of the sulfhydryl analogs are significantly more potent as inhibitors than the corresponding compounds in which the SH group is replaced by an H atom.     

Stimulation of gamma-aminobutyric acid synthesis activity in brown rice by a chitosan/glutamic acid germination solution and calcium/calmodulin

Changes in the concentrations of gamma-aminobutyric acid (GABA), soluble calcium ions, glutamic acid, and the activity of glutamate decarboxylase (GAD) were investigated in non-germinated vs. germinated brown rice. Brown rice was germinated for 72 h by applying each of the following solutions: (1) distilled water, (2) 5 mM lactic acid, (3) 50 ppm chitosan in 5 mM lactic acid, (4) 5 mM glutamic acid, and (5) 50 ppm chitosan in 5 mM glutamic acid. GABA concentrations were enhanced in all of the germinated brown rice when compared to the non-germinated brown rice. The GABA concentration was highest in the chitosan/glutamic acid that germinated brown rice at 2,011 nmol/g fresh weight, which was 13 times higher than the GABA concentration in the non-germinated brown rice at 154 nmol/g fresh weight. The concentrations of glutamic acid were significantly decreased in all of the germinated rice, regardless of the germination solution. Soluble calcium and GAD were higher in the germinated brown rice with the chitosan/glutamic acid solution when compared to the rice that was germinated in the other solutions. GAD that was partially purified from germinated brown rice was stimulated about 3.6-fold by the addition of calmodulin in the presence of calcium. These data show that the germination of brown rice in a chitosan/glutamic acid solution can significantly increase GABA synthesis activity and the concentration of GABA.     

An electrostatic model of a membrane ion pump

The model is based upon an ion channel with an electric dipolar structure. With simplifying assumptions it is possible to calculate that a typical channel, 1 nm in diameter and 5 nm long, could contain at most two or three univalent cations at a time. The channel ion binding sites have an effective affinity for ions from the fluid bathing the negative end of the channel, several orders of magnitude higher than their affinity for ions from the fluid bathing the positive end of the channel. The approach of an external, positively charged body to the negative end of the channel, is sufficient to convert the two- or three-channel ion sites with high affinity for ions from the fluid bathing this end into very low affinity sites for the same ions that now have access only to the fluid bathing the other end of the channel. The change in affinity and fluid access requires no molecular or electrical change in the channel structure other than the passive superposition of the electrostatic potential of the dipolar channel and that of the charged body. An oscillating electric field externally applied to an electric dipolar channel is shown to result in the unidirectional pumping of cations in the direction of the channel dipole even against large adverse ion concentration gradients. The energy required must be supplied by the sources of the electric field. By using two such channels in close proximity, one selective for K+ ions with its dipole moment pointing into a cell and the other selective for Na+ ions with its dipole moment pointing out from the cell, it is possible to construct a model pump with calculated properties that simulate many of those measured for Na+-K+-ATPase, with both physiological and artificial ionic concentrations.     

Anti-HIV activity of a series of cosalane amino acid conjugates

The binding of the anti-HIV agent cosalane to CD4 is thought to involve ionic interactions of negatively charged carboxylates of the ligand with positively charged residues on the surface of the protein. The purpose of the present study was to examine the hypothesis that the two carboxyl groups of cosalane could be sacrificed through conjugation to amino acids, and the anti-HIV activity still be retained, provided that at least two new carboxyl groups are contributed by the amino acid residues.     

Effect of aliphatic alcohols on the conformation of poly (l-ornithine hydrobromide) as studied by square-pulse and reversing-pulse electric birefringence

The electric birefringence and circular dichroism spectra of poly(l-ornithine hydrobromide) have been measured in ethanol/water, 2-propanol/water and tertiary butyl alcohol/water mixtures of various compositions. This charged polypeptide underwent a transition from the coil conformation to the helical conformation at high alcohol content in every case tested. Anomalous birefringence signals, indicative of a field-induced helix-to-coil transition. were observed at high electric fields only in the case of ethanol/water mixtures. The reversing-pulse electric birefringence of this polypeptide has been studied in ethanol/water mixtures and in neutral aqueous solution. Upon rapid reversal of the pulse field, no transient could be observed. This confirms that the electric-field orientation of poly(l-ornithine hydrobromide) results predominantly from the contribution of the counterion-induced dipole moment, regardless of its molecular conformations. It is very probable that the backbone permanent dipole moment of the helical conformation is largely suppressed by the counterion-induced dipole moment in the ionized form.     

Calcium binding by human erythrocyte membranes. Significance of carboxyl, amino and thiol groups

1. The role of the ionized carboxyl groups of proteins of the erythrocyte membrane as Ca(2+) receptor sites was investigated. A water-soluble carbodi-imide [1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide methotoluene-p-sulphonate], referred to as carbodi-imide reagent, and glycine methyl ester were used to modify the free carboxyl groups of the membrane. The degree of modification was estimated from amino acid analyses, which showed the amount of glycine incorporated. As the concentration of carbodi-imide reagent was raised (0.1-0.4m) incorporation of glycine increased and Ca(2+) binding decreased by about 77%. At 0.4m-carbodi-imide reagent all of the binding of Ca(2+) to protein was abolished and it was estimated that about 37% of the side-chain carboxyl groups of aspartic acid plus glutamic acid had been blocked by glycine. 2. Acetylation of all of the free amino groups was achieved by incubating the erythrocyte ;ghosts' at pH10.3 with acetic anhydride (10-15mg/10mg of ;ghost' protein). Acetylation increased by 1.5-fold the capacity of the ;ghost' to bind Ca(2+), indicating that the remaining carboxyl groups of aspartic acid and glutamic acid were made available for Ca(2+) binding by this procedure. These findings support the concept that in normal ;ghosts', at pH7.4, Ca(2+) binding to free carboxyl groups is partially hindered by the presence of charged amino groups. 3. Treatment of ;ghosts' with N-acetylneuraminidase, which removed 94% of sialic acid residues, and treatment with 1mm-p-chloromercuribenzoate did not alter Ca(2+) binding. The major effect of 5.8mm-p-chloromercuribenzoate upon ;ghosts' was to cause a solubilization of a calcium-membrane complex, which included about one-third of the ;ghost' protein. The molar ratio of Ca(2+): protein in the solubilized material was the same as that in the intact (untreated) ;ghosts'.     

Electrostatic field effects on membrane domain segregation and on lateral diffusion

Natural membranes are organized structures of neutral and charged molecules bearing dipole moments which generate local non-homogeneous electric fields. When subjected to such fields, the molecules experience net forces that can modify the lipid and protein organization, thus modulating cell activities and influencing (or even dominating) the biological functions. The energetics of electrostatic interactions in membranes is a long-range effect which can vary over distance within r⁻¹ to r⁻³. In the case of a dipole interacting with a plane of dipoles, e.g. a protein interacting with a lipid domain, the interaction is stronger than two punctual dipoles and depends on the size of the domain. In this article, we review several contributions on how electrostatic interactions in the membrane plane can modulate the phase behavior, surface topography and mechanical properties in monolayers and bilayers.     

Two amino acids in each of D1 and D2 dopamine receptor cytoplasmic regions are involved in D1-D2 heteromer formation

D(1) and D(2) dopamine receptors exist as heteromers in cells and brain tissue and are dynamically regulated and separated by agonist concentrations at the cell surface. We determined that these receptor pairs interact primarily through discrete amino acids in the cytoplasmic regions of each receptor, with no evidence of any D(1)-D(2) receptor transmembrane interaction found. Specifically involved in heteromer formation we identified, in intracellular loop 3 of the D(2) receptor, two adjacent arginine residues. Substitution of one of the arginine pair prevented heteromer formation. Also involved in heteromer formation we identified, in the carboxyl tail of the D(1) receptor, two adjacent glutamic acid residues. Substitution of one of the glutamic acid pair prevented heteromer formation. These amino acid pairs in D(1) and D(2) receptors are oppositely charged, and presumably interact directly by electrostatic interactions.