Role of electrostatic interactions in cohesion of bacterial biofilms

Significant decreases in the apparent viscosity of a bacterial biofilm suspension were measured following addition of sodium, potassium, magnesium, or calcium salts, whereas iron salts increased the viscosity. Electrostatic interactions contribute to biofilm cohesion and iron cations are potent crosslinkers of the biofilm matrix.     

Role of electrostatic interactions in PDZ domain ligand recognition

PDZ domains are protein-protein interaction modules that normally recognize short C-terminal peptides. The apparent requirement for a ligand with a free terminal carboxylate group has led to the proposal that electrostatic interactions with the terminus play a significant role in recognition. However, this model has been called into question by the more recent finding that PDZ domains can recognize some internal peptide motifs that occur within a specific secondary structure context. Although these motifs bind at the same interface, they lack a terminal charge. Here we have investigated the role of electrostatics in PDZ-mediated recognition in the mouse alpha1-syntrophin PDZ domain by examining the salt dependence of binding to both terminal and internal ligands and the effects of mutating a conserved basic residue previously proposed to play a role in electrostatic recognition. These studies indicate that direct electrostatic interactions with the peptide terminus do not play a significant energetic role in binding. Additional chemical modification studies of the peptide terminus support a model in which steric and hydrogen bonding complementarity play a primary role in recognition specificity. Peptides with a free carboxy terminus, or presented within a specific structural context, can satisfy these requirements.     

Vulnerability of keto bile acids to alkaline hydrolysis.

Rigorous alkaline hydrolysis of the two primary (cholic and chenodeoxycholic) and of the two preponderant secondary (deoxycholic and lithocholic) bile acids found in bile led to excellent recoveries. Such was not the case with 11 different keto bile acid standards. Recoveries for a number of standards were unacceptably low and a variety of artefactural products were tentatively identified by gas-liquid chromatography. Keto bile acids bearing a keto gropu on C-3 were particularly vulnerable. In view of thee findings, quantitative and qualitative data reported on biological specimens submitted to saponification in ethanol, methanol, or even in water are of questionable significance.     

Role of erythrocytes in leukocyte-endothelial interactions: mathematical model and experimental validation.

The binding of circulating cells to the vascular wall is a central process in inflammation, metastasis, and therapeutic cell delivery. Previous in vitro studies have identified the adhesion molecules on various circulating cells and the endothelium that govern the process under static conditions. Other studies have attempted to simulate in vivo conditions by subjecting adherent cells to shear stress as they interact with the endothelial cells in vitro. These experiments are generally performed with the cells suspended in Newtonian solutions. However, in vivo conditions are more complex because of the non-Newtonian flow of blood, which is a suspension consisting of 20-40% erythrocytes by volume. The forces imparted by the erythrocytes in the flow can contribute to the process of cell adhesion. A number of experimental and theoretical studies have suggested that the rheology of blood can influence the binding of circulating leukocytes by increasing the normal and axial forces on leukocytes or the frequency of their collision with the vessel wall, but there have been no systematic investigations of these phenomena to date. The present study quantifies the contribution of red blood cells (RBCs) in cell capture and adhesion to endothelial monolayers using a combination of mathematical modeling and in vitro studies. Mathematical modeling of the flow experiments suggested a physical mechanism involving RBC-induced leukocyte dispersion and/or increased normal adhesive contact. Flow chamber studies performed with and without RBCs in the suspending medium showed increases in wall collision and binding frequencies, and a decrease in rolling velocity in the presence of erythrocytes. Increased fluid viscosity alone did not influence the binding frequency, and the differences could not be attributed to large near-wall excesses of the lymphocytes. The results indicate that RBCs aid in the transport and initial engagement of lymphocytes to the vascular wall, modifying the existing paradigm for immune cell surveillance of the vascular endothelium by adding the erythrocyte as an essential contributor to this process.     

Introduction of aliphatic amino and hydroxy groups to keto steroids using O-substituted hydroxylamines.

(Aminooxy)butyl- and -hexylamines and alcohols were synthesized by the Ing-Manske modification of the Gabriel synthesis. The aminooxy group of these heterobifunctional spacer reagents is a far more powerful nucleophile than the amino or hydroxy group because of the oxygen atom adjacent to the amino group (alpha-effect). The aminooxy group reacts readily with keto groups while the amino or hydroxy (or other) group remains free for further reactions. These excellent heterobifunctional spacer reagents were used here to derivatize keto steroids in an alkaline alcoholic solution. Using the described, general, and easy one-step synthesis, aliphatic amino or hydroxy groups with a spacer arm have been introduced to testosterone, 6-ketoestradiol, and cortisol.     

The electrostatic contribution to interactions of some enzymes with polyelectrolytes

To explain the inhibitory action of polyelectrolytes on enzymes and, in particular, to define potentially reactive zones for the binding of polyelectrolyte, the electric potential of enzymes lactate dehydrogenase and glutamate dehydrogenase was calculated using the solution of the Poisson-Boltzmann equation by a numerical method with the use of the Gauss-Seidel relaxation method at three pH values: 6.5, 7.0, and 8.0 and three values of ionic strength: 50, 100, and 150 mm. On the basis of these calculations and their visualization, representative sites for favorable binding of polyanions were determined as extended areas on the surface of proteins with the positive potential in the neutral pH region. It was shown that there is a correlation between the area of positive potential and the efficiency of enzyme inactivation for a number of pH values and concentrations of salt for two enzymes. The calculations performed allowed one to explain the inhibitory action of polyelectrolytes on the specified enzymes to understand the difference between the values of polyelectrolyte inactivation constants for the two enzymes and estimate the minimal areas of the positive potential on the protein surface that provide their effective inhibition.     

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.     

Role of electrostatic interactions in 2,2,2-trifluoroethanol-induced structural changes and aggregation of alpha-chymotrypsin

It has been recently demonstrated that alpha-chymotrypsin (CT) can be driven toward amyloid aggregation by addition of 2,2,2-trifluoroethanol (TFE), at intermediate concentrations. In the present article, the process of TFE-induced CT aggregation was investigated in more detailed kinetic terms where the effects of medium conditions, such as temperature, presence of kosmotropic and chaotropic salts, pH and chemical modification of lysine residues were examined. Various techniques, including light scattering, fluorescence and circular dichroism spectroscopy, were used to follow and characterize this process. The kinetics of aggregation was found to obey a second-order reaction with respect to protein concentration. The aggregation-prone A-state and aggregation-deficient TFE- or T-state of CT were found to be induced at lower TFE concentrations in the presence of salts. Use of acidic and alkaline conditions and lysine modification also promoted the formation of the T-state. Results presented suggest a role for electrostatic interactions in the aggregation process.     

Structures of the contryphan family of cyclic peptides. Role of electrostatic interactions in cis-trans isomerism

The contryphan family of cyclic peptides, isolated recently from various species of cone shell, has the conserved sequence motif NH(3)(+)-X(1)COD-WX(5)PWC-NH(2), where X(1) is either Gly or absent, O is 4-trans-hydroxyproline, and X(5) is Glu, Asp, or Gln. The solution structures described herein of two new naturally occurring contryphan sequences, contryphan-Sm and des[Gly1]-contryphan-R, are similar to those of contryphan-R, the structure of which has been determined recently [Pallaghy et al. (1999) Biochemistry 38, 11553-11559]. The (1)H NMR chemical shifts of another naturally occurring peptide, contryphan-P, indicate that it also adopts a similar structure. All of these contryphans exist in solution as a mixture of two conformers due to cis-trans isomerization about the Cys2-Hyp3 peptide bond. The lower cis-trans ratio for contryphan-Sm enabled elucidation of the 3D structure of both its major and its minor forms, for which the patterns of (3)J(H)(alpha)(HN) coupling constants are very different. As with contryphan-R, the structure of the major form of contryphan-Sm (cis Cys2-Hyp3 peptide bond) contains an N-terminal chain reversal and a C-terminal type I beta-turn. The minor conformer (trans peptide bond) forms a hairpin structure with sheetlike hydrogen bonds and a type II beta-turn, with the D-Trp4 at the 'Gly position' of the turn. The ratio of conformers arising from cis-trans isomerism around the peptide bond preceding Hyp3 is sensitive to both the amino acid sequence and the solution conditions, varying from 2.7:1 to 17:1 across the five sequences. The sequence and structural determinants of the cis-trans isomerism have been elucidated by comparison of the cis-trans ratios for these peptides with those for contryphan-R and an N-acetylated derivative thereof. The cis-trans ratio is reduced for peptides in which either the charged N-terminal ammonium or the X(5) side-chain carboxylate is neutralized, implying that an electrostatic interaction between these groups stabilizes the cis conformer relative to the trans. These results on the structures and cis-trans equilibrium of different conformers suggest a paradigm of 'locally determined but globally selected' folding for cyclic peptides and constrained protein loops, where the series of stereochemical centers in the loop dictates the favorable conformations and the equilibrium is determined by a small number of side-chain interactions.     

The role of electrostatic forces in pollination

This paper reviews research on the role of electrostatic forces in pollination, both in natural and in agricultural systems. Researchers from various fields of biological studies have reported phenomena which they related to electrostatic forces. The theory of electrostatically mediated pollen transfer between insect pollinators and the flowers they visit is described, including recent studies which confirmed that the accumulated charges on airborne honey bees are sufficient for non-contact pollen detachment by electrostatic forces (i.e., electrostatic pollination). The most important morphological features in flower adaptiveness to electrostatic pollination were determined by means of two theoretical models of a flower exposed to an approaching charged cloud of pollen; they are style length and flower opening. Supplementary pollination by using electrostatic techniques is reported, and its possible importance in modern agriculture is discussed.     

The optimization of protein-solvent interactions: thermostability and the role of hydrophobic and electrostatic interactions.

Protein-solvent interactions were analyzed using an optimization parameter based on the ratio of the solvent-accessible area in the native and the unfolded protein structure. The calculations were performed for a set of 183 nonhomologous proteins with known three-dimensional structure available in the Protein Data Bank. The dependence of the total solvent-accessible surface area on the protein molecular mass was analyzed. It was shown that there is no difference between the monomeric and oligomeric proteins with respect to the solvent-accessible area. The results also suggested that for proteins with molecular mass above some critical mass, which is about 28 kDa, a formation of domain structure or subunit aggregation into oligomers is preferred rather than a further enlargement of a single domain structure. An analysis of the optimization of both protein-solvent and charge-charge interactions was performed for 14 proteins from thermophilic organisms. The comparison of the optimization parameters calculated for proteins from thermophiles and mesophiles showed that the former are generally characterized by a high degree of optimization of the hydrophobic interactions or, in cases where the optimization of the hydrophobic interactions is not sufficiently high, by highly optimized charge-charge interactions.     

Role of the protein side-chain fluctuations on the strength of pair-wise electrostatic interactions: comparing experimental with computed pK(a)s

The effect of the protein side-chain fluctuations on the strength of electrostatic interactions was studied. The effect was modeled on 7 different crystal structures on the same enzyme as well as on 20 molecular dynamics snapshot structures. It was shown that the side-chain flexibility affects predominantly the magnitude of the strong pair-wise interactions, that is, the pair-wise interaction among ion pairs, and practically does not affect the interactions with the rest of the protein. This was used to suggest a correction function that should be applied to the original pair-wise electrostatic interaction to mimic the effects of the fluctuations. The procedure is applied on three ion pairs identified in lysozyme. It was shown that sampling different side-chain rotamers and modifying the strength of the pair-wise interaction energies makes calculated pK(a)s less sensitive to the fluctuations of the structure and improves the prediction accuracy.     

Contributions of long-range electrostatic interactions to 4-chlorobenzoyl-CoA dehalogenase catalysis: a combined theoretical and experimental study

It is well established that electrostatic interactions play a vital role in enzyme catalysis. In this work, we report theory-guided mutation experiments that identified strong electrostatic contributions of a remote residue, namely, Glu232 located on the adjacent subunit, to 4-chlorobenzoyl-CoA dehalogenase catalysis. The Glu232Asp mutant was found to bind the substrate analogue 4-methylbenzoyl-CoA more tightly than does the wild-type dehalogenase. In contrast, the kcat for 4-chlorobenzoyl-CoA conversion to product was reduced 10000-fold in the mutant. UV difference spectra measured for the respective enzyme-ligand complexes revealed an approximately 3-fold shift in the equilibrium of the two active site conformers away from that inducing strong pi-electron polarization in the ligand benzoyl ring. Increased substrate binding, decreased ring polarization, and decreased catalytic efficiency indicated that the repositioning of the point charge in the Glu232Asp mutant might affect the orientation of the Asp145 carboxylate with respect to the substrate aromatic ring. The time course for formation and reaction of the arylated enzyme intermediate during a single turnover was measured for wild-type and Glu232Asp mutant dehalogenases. The accumulation of arylated enzyme in the wild-type dehalogenase was not observed in the mutant. This indicates that the reduced turnover rate in the mutant is the result of a slow arylation of Asp145, owing to decreased efficiency in substrate nucleophilic attack by Asp145. To rationalize the experimental observations, a theoretical model is proposed, which computes the potential of mean force for the nucleophilic aromatic substitution step using a hybrid quantum mechanical/molecular mechanical method. To this end, the removal or reorientation of the side chain charge of residue 232, modeled respectively by the Glu232Gln and Glu232Asp mutants, is shown to increase the rate-limiting energy barrier. The calculated 23.1 kcal/mol free energy barrier for formation of the Meisenheimer intermediate in the Glu232Asp mutant represents an increase of 6 kcal/mol relative to that of the wild-type enzyme, consistent with the 5.6 kcal/mol increase calculated from the difference in experimentally determined rate constants. On the basis of the combination of the experimental and theoretical evidence, we hypothesize that the Glu232(B) residue contributes to catalysis by providing an electrostatic force that acts on the Asp145 nucleophile.     

Role of electrostatic interactions for the stability and folding behavior of cold shock protein

The impacts of three charged‐residue‐involved mutations, E46A, R3E, and R3E/L66E, on the thermostability and folding behavior of the cold shock protein from the themophile Bacillus caldolyticus (Bc‐Csp) were investigated by using a modified Gō‐like model, in which the nonspecific electrostatic interactions of charged residues were taken into account. Our simulation results show that the wild‐type Bc‐Csp and its three mutants are all two‐sate folders, which is consistent with the experimental observations. It is found that these three mutations all lead to a decrease of protein thermodynamical stability, and the effect of R3E mutation is the strongest. The lower stability of these three mutants is due to the increase of the enthalpy of the folded state and the entropy of the unfolded state. Using this model, we also studied the folding kinetics and the folding/unfolding pathway of the wild‐type Bc‐Csp as well as its three mutants and then discussed the effects of electrostatic interactions on the folding kinetics. The results indicate that the substitutions at positions 3 and 46 largely decrease the folding kinetics, whereas the mutation of residue 66 only slightly decreases the folding rate. This result agrees well with the experimental observations. It is also found that these mutations have little effects on the folding transition state and the folding pathway, in which the N‐terminal β sheet folds earlier than the C‐terminal region. We also investigated the detailed unfolding pathway and found that it is really the reverse of the folding pathway, providing the validity of our simulation results. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Role of the electrostatic interactions in pre-orientation of subunits in the formation of protein-protein complexes

Theoretical estimation of contribution of the electrostatic interactions to pre-orientation of ribonuclease subunits in process of complex formation was carried out. The subunit was considered as a multipole consisting of partial charges of all atoms of the molecule. The object of investigation was a system of two subunits with their centers of gravity fixed at some distance in vacuum. It was proposed that each subunit independently could rotate freely around its fixed center of gravity. The relative orientation states of the subunits in such system were searched at which the system has electrostatic energy minima (equilibrium states). In first approximation the equilibrium states were found using especially designed approximate method for electrostatic interaction energy calculation, which permitted to calculate and compare the energies of the system in 24(5) (approximately 8 10(6)) states with different mutual orientation of subunits. The angular coordinates of the found equilibrium states were further specified by calculation with gradient sliding method. Angular coordinates of the equilibrium states and the shapes of energy surface cuts along each coordinate angle were calculated also for the intersubunits distances diminished down to 50 angstroms. The dispersions of the angular coordinates of equilibrium states caused by heat movement (at T=300 degrees) and their changes with shortening the distance between centers of gravity of subunits were estimated. Mutual orientation of subunits in the equilibrium states of the system under consideration was found to be similar to their mutual orientations in complex. Also it was found that relaxation time of the system, caused by electrostatic interaction of subunits, after removing the system from an equilibrium state, is much less in vacuum than the mean time between their Brownian collisions at room temperature. It follows from these results that in the case of ribonuclease in vacuum the electrostatic interactions of its subunits must be strong enough to realize the effective pre-orientation of subunits during their Brownian approach from distances of the order 100 angstroms. Preliminary consideration taking into account the effect of surrounding water molecules on the electrostatic interactions of ribonuclease subunits showed that weakening of the interaction must be much less than in the case when one uses in its calculation the macroscopic dielectric permeability value equal to 80. So the results obtained for vacuum seem to be true for water solution also. More strict theoretical analysis of this problem will be carried out in the following publication.