pH dependence of electrostatic interaction between ion-penetrable membranes

A theory on the electrostatic repulsion between ion-penetrable membranes proposed previously by us is extended by taking into account the degree of dissociation of the membrane-fixed ionizable groups. A system of equations which determines the pH dependence of the membrane interaction is presented. The density of membrane-fixed charges is consistently determined as a function of the electric potential so that both the membrane-fixed charge density and the potential are not constant but functions of the membrane separation. The pH at the surface of interacting membranes is also calculated as a function of the membrane separation.     

Electrostatic interaction between gating charges in nerve membranes

The electrostatic interaction between charges within a nerve membrane is considered. It is seen that the electrostatic repulsion between so called gating particles of an ionic channel cannot be neglected in the formulation of excitability models based on gating charges. Some general conclusions are drawn about different models for nerve membrane excitability.     

Electrostatic interaction between merocyanine 540 and liposomal and mitochondrial membranes

Summary The fluorescence of merocyanine 540 (MC) in liposomal and mitochondrial suspensions was measured under various conditions. Under a given condition, both the amount of dye bound to the membrane and the zeta potential were determined simultaneously. It was found that the fluorescence intensity was proportional to the amount of bound dye and correlated with the zeta potential of particles. The fluorescence intensity was represented quantitatively in terms of the Langmuir adsorption isotherm, when the electrostatic interaction acting between MC and membrane surface was properly taken into account. It was concluded that the changes in MC fluorescence in the liposomal and mitochondrial suspensions are mainly attributed to the changes in the surface potential of the membranes.     

Electrostatic interaction between two charged spherical molecules

An explicit analytic expression is obtained for the electrostatic energy of the interaction between two ion-impenetrable space-charged hard spheres as a model for spherical molecules in an electrolyte solution on the basis of the linearized Poisson-Boltzmann equation. An explicit expression for the potential distribution in a 3D-space is also found. The polarization effects due to the mutual influence between the spheres are taken into account. The analysis is done by assuming different dielectric permittivities of the respective spheres and of the solution as well. It is shown that the correction terms in the expression for the total energy of interaction arising from the polarization effects always correspond to forces of attraction between the spheres. The contribution of these terms to the total energy of interaction depends on the distance between the two spheres and the dielectric permittivities of the spheres and the solution as well as on the electrolyte concentration in the solution. A numerical simulation of the potential field topography is carried out at several values of the Debye-Hückel parameter. It is shown that the polarization effect can produce significant changes in the potential distribution in the case of strong interacting spheres.     

Electrostatic interaction between redox cofactors in photosynthetic reaction centers

Intramolecular electron transfer within proteins is an essential process in bioenergetics. Redox cofactors are embedded in proteins, and this matrix strongly influences their redox potential. Several cofactors are usually found in these complexes, and they are structurally organized in a chain with distances between the electron donor and acceptor short enough to allow rapid electron tunneling. Among the different interactions that contribute to the determination of the redox potential of these cofactors, electrostatic interactions are important but restive to direct experimental characterization. The influence of interaction between cofactors is evidenced here experimentally by means of redox titrations and time-resolved spectroscopy in a chimeric bacterial reaction center (Maki, H., Matsuura, K., Shimada, K., and Nagashima, K. V. P. (2003) J. Biol. Chem. 278, 3921-3928) composed of the core subunits of Rubrivivax gelatinosus and the tetraheme cytochrome of Blastochloris viridis. The absorption spectra and orientations of the various cofactors of this chimeric reaction center are similar to those found in their respective native protein, indicating that their local environment is conserved. However, the redox potentials of both the primary electron donor and its closest heme are changed. The redox potential of the primary electron donor is downshifted in the chimeric reaction center when compared with the wild type, whereas, conversely, that of its closet heme is upshifted. We propose a model in which these reciprocal shifts in the midpoint potentials of two electron transfer partners are explained by an electrostatic interaction between them.     

Electrostatic interaction in the reaction between porphyrin and DNA

The association of tetrakis(4-N-methylpyridyl)porphine with salmon testes DNA in solution has been investigated in the presence of ethanol at 25 degrees C. It was found that the association constant was 1.14 x 10(6) M-1 and it increased with increasing the mole fraction of added ethanol. The behavior was very different from that in the case of usual intercalaters.     

Electrostatic and hydrophobic interactions governing the interaction and binding of beta-lactoglobulin to membranes

The role of electrostatic and hydrophobic interactions in the binding and penetration of beta-lactoglobulin (betaLG) to preformed lipid membranes was studied using various phospholipid micelles and vesicles. Zwitterionic lysophospholipid micelles are able to induce the beta-sheet to alpha-helix transition, as judged by circular dichroism (CD), but the degree of transition is dramatically below and the amount of lipid required above that for anionic phospholipids with equivalent hydrocarbon chains. Anionic phospholipids with short hydrocarbon chains induce only low alpha-helical content in betaLG as compared to phospholipids with the same head group but longer hydrocarbon chains. These results suggest that both electrostatic and hydrophobic interactions are indispensable in betaLG-lipid interaction. Furthermore, air-water interface monolayer surface pressure and fluorescence anisotropy studies reveal that the membrane insertion of betaLG strongly depends on the nature of phospholipids, given the identical headgroup, particularly lipid packing. These results are supported by urea denaturation and acrylamide fluorescence quenching tests and by the FTIR-ATR polarization results for betaLG in multilayers on a surface. Under the same experimental conditions, the membrane binding and insertion of betaLG as well as the stability of the betaLG-lipid complexes can be enhanced by lowering the pH. Collectively, electrostatic interactions play a crucial role in all the processes involved in the betaLG-lipid interaction, while the presence of hydrophobic interaction remains necessary. Finally, betaLG biological function in the transport of fatty acids was tested by demonstrating the release of 2-AS from a 2-AS-betaLG complex on binding to lipids.     

Electrostatic recognition between enzyme and inhibitor: interaction between papain and leupeptin

Electrostatic forces are involved in a wide variety of molecular interactions that are of biological interest, including, among others, DNA-Protein interactions, protein folding, and the interactions between enzymes and their substrates and inhibitors. In this work, the interaction between papain and an inhibitor, leupeptin, is analyzed from the point of view of their electrostatic interaction. The computations enable one to suggest that negatively charged amino acids located in the region of the active site are responsible for creating an environment that enables efficient binding of the inhibitor. This binding occurs despite the fact that the net global charge of both molecules is positive; an explanation for this apparent contradiction is proposed.     

Relationship among the surface potential, Donnan potential and charge density of ion-penetrable membranes

Calculation of the potential distribution across a uniformly charged ion-penetrable membrane that we developed previously is extended to derive a relationship among the surface potential, Donnan potential and charge density of an ion-penetrable membrane in a mixed solution of 2-1 and 1-1 electrolytes. We also present an approximate method for calculating the surface potential/Donnan potential/charge density relationship for membranes with an arbitrary distribution of membrane-fixed charges.     

Lipophilic interaction between thylakoid membranes and aliphatic compounds

Spinach chloroplasts pre-incubated at various temperatures, changed their photosynthetic activities (measured at 15°C) as the incubation temperature was raised; these activities showed characteristic activitytemperature profiles as follows:

1. The profiles were shifted to lower temperatures if various aliphatic compounds were present in the incubation mixture.
2. In general, the extent of the shift was proportional to the product of the concentration and the partition coefficient of the compound, i.e. to the concentration of the compound partitioned in the lipophilic chloroplast phase.

The combined effects of heat and the aliphatic compounds tested indicated that the lipophilic groups in these compounds play a determinative role in the heat denaturation of thylakoids. The consequence of such structure alterations is inactivation of photosynthetic functions. The lipophilic properties of the spinach thylakoid membrane as compared with certain artificial and natural membranes are described.     

alpha-Synuclein exhibits competitive interaction between calmodulin and synthetic membranes

alpha-Synuclein, a pathological component of Parkinson's disease by constituting the Lewy bodies, has been suggested to be involved in membrane biogenesis via induction of amphipathic alpha-helices. Since the amphipathic alpha-helix is also known as a recognition signal of calmodulin for its target proteins, molecular interaction between alpha-synuclein and calmodulin has been investigated. By employing a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, alpha-synuclein has been shown to yield a heterodimeric 1 : 1 complex with calmodulin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and even absence of calcium, whereas beta-synuclein was more dependent upon calcium for its calmodulin interaction. The selective calmodulin interaction of alpha-synuclein in the absence of calcium was also demonstrated with the aggregation kinetics of the synucleins in which only the alpha-synuclein aggregation was affected by calmodulin. A reversible binding assay confirmed that alpha-synuclein interacted with the Ca2+-free as well as the Ca2+-bound calmodulins with almost identical Kds of 0.35 micro m and 0.31 micro m, respectively, while beta-synuclein preferentially recognized the Ca2+-bound form with a Kd of 0.68 micro m. By using a C-terminally truncated alpha-synuclein of alpha-syn97, the calmodulin binding site(s) on alpha-synuclein was(were) shown to be located on the N-terminal region where the amphipathic alpha-helices have been suggested to be induced upon membrane interaction. By employing liposome and calmodulin in a state of being either soluble or immobilized on agarose, actual competition of alpha-synuclein between membranes and calmodulin was demonstrated with the observation that alpha-synuclein previously bound to the liposome was released upon specific interaction with the calmodulins. Taken together, these data may suggest that alpha-synuclein could act not only as a negative regulator for calmodulin in the presence and even absence of calcium, but it could also exert its activity at the interface between calmodulin and membranes.     

Electrostatic interaction and complex formation between gum arabic and bovine serum albumin

The interaction of gum arabic (GA) and bovine serum albumin (BSA) has been investigated through turbidity and light scattering intensity measurements and by the use of dynamic light scattering, laser Doppler velocimetry, and isothermal titration calorimetry. It has been shown that GA and BSA can form soluble and insoluble complexes depending on the solution pH and the mixing ratio and is a function of the net charge on the complex. Soluble complexes were obtained when the electrophoretic mobility was greater than ±1. 5 μm s(-1) V(-1) cm(-1). Changes in the value of the isoelectric point of the complexes with mixing ratio and isothermal titration calorimetric data indicated that complexes formed at pHs 3 and 4 consisted of ～60 BSA molecules for every GA molecule, while at pH 5 there were ～10 BSA molecules per GA molecule. Calorimetric studies also indicated that the interaction occurred in two stages at both pH 3 and pH 4, but that the nature of the interaction at these two pH values was significantly different. This was attributed to differences in the relative magnitude of the positive and negative charges on the BSA and GA, respectively, and possibly due to changes in the BSA conformation. The fact that there is an interaction at pH 5, which is above the isoelectric point of the BSA, is due to the interaction of the carboxylate groups on the GA with positive patches on the BSA or to the charge regulation of the protein-polysaccharide system brought about by changes in dissociation equilibria. Complexation is reduced as the ionic strength of the solvent increases and is prevented at a NaCl concentration of 120 mM.     

Electrostatic dependence of the thrombin-thrombomodulin interaction

The rate constants for the binding interaction between thrombin and a fully active fragment of its anticoagulant cofactor, thrombomodulin, have been determined by surface plasmon resonance. At physiological ionic strength, the k(a) was 6.7x10(6) M(-1) s(-1 )and the dissociation rate constant was 0.033 s(-1). These extremely fast association and dissociation rates resulted in an overall binding equilibrium constant of 4.9 nM, which is similar to previously reported values. Changing the ionic strength from 100 mM to 250 mM NaCl caused a tenfold decrease in the association rate while the dissociation rate did not change significantly. A similar effect was observed with tetramethylammonium chloride. A Debye-Hückel plot of the data had a slope of -6 and an intercept at 0 ionic strength of 10(9) M(-1) s(-1). The same slope and intercept were obtained for data that was collected in the presence of glycerol to slow the association rates. These results show that the thrombin-TM456 interaction is extremely rapid and nearly completely electrostatically steered. An association model is presented in which TM456 approaches thrombin along the direction of the thrombin molecular dipole.     

Electrostatic interaction in atomic force microscopy

In atomic force microscopy, the stylus experiences an electrostatic force when imaging in aqueous medium above a charged surface. This force has been calculated numerically with continuum theory for a silicon nitrite or silicon oxide stylus. For comparison, the Van der Waals force was also calculated. In contrast to the Van der Waals attraction, the electrostatic force is repulsive. At a distance of 0.5 nm the electrostatic force is typically 10^-12-10^-10 N and thus comparable in strength to the Van der Waals force. The electrostatic force increases with increasing surface charge density and decreases roughly exponentially with distance. It can be reduced by imaging in high salt concentrations. Below surface potentials of ≈50 mV, a simple analytical approximation of the electrostatic force is described.     

Electrostatic interaction between charybdotoxin and a tetrameric mutant of Shaker K(+) channels

The scorpion toxin, Charybdotoxin (CTX), blocks homotetrameric, voltage-gated K(+) channels by binding near the outer entrance to the pore in one of four indistinguishable orientations. We have determined the pH-dependence of CTX block of a tetrameric Shaker potassium channel with a single copy of a histidine replacing the wild-type phenylalanine at position 425. We compared this pH-dependence with that from homotetrameric channels with four copies of the mutation. We found that protonation of a single amino acid at position 425 had a large effect on the affinity of the channel for CTX-much larger than expected if only one of the four CTX binding orientations was disrupted. The pK(a) for the H(+)-ion induced protection from CTX block indicates that the electrostatic environment near position 425 is likely basic in nature, perhaps because of the proximity of lysine 427. We also examined the pH-dependence of block of channels with one and four copies of the histidine mutation by CTX containing neutralizing mutations of four basic residues on the active face of the toxin. The results suggested an orientation of CTX on the channel that places three of the positively charged CTX residues very near three of the four Shaker 425 positions.     

A novel mechanism of interaction between alpha-synuclein and biological membranes

Conformational abnormalities and aggregation of alpha-synuclein (alpha-syn) have been linked to the pathogenesis of Parkinson's (PD) and related diseases. It has been shown that alpha-syn can stably bind artificial phospholipid vesicles through alpha-helix formation in its N-terminal repeat region. However, little is known about the membrane interaction in cells. In the current study, we determined the membrane-binding properties of alpha-syn to biological membranes by using bi-functional chemical crosslinkers, which allow the detection of transient, but specific, interactions. By utilizing various point mutations and deletions within alpha-syn, we demonstrated that the membrane interaction of alpha-syn in cells is also mediated by alpha-helix formation in the N-terminal repeat region. Moreover, the PD-linked A30P mutation causes reduced membrane binding, which is concordant with the artificial membrane studies. However, contrary to the interaction with artificial membranes, the interaction with biological membranes is rapidly reversible and is not driven by electrostatic attraction. Furthermore, the interaction of alpha-syn with cellular membranes occurs only in the presence of non-protein and non-lipid cytosolic components, which distinguishes it from the spontaneity of the interaction with artificial membranes. More interestingly, addition of the cytosolic preparation to artificial membranes resulted in the transient, charge-independent binding of alpha-syn similar to the interaction with biological membranes. These results suggest that in cells, alpha-syn is engaged in a fundamentally different mode of membrane interaction than the charge-dependent artificial membrane binding, and the mode of interaction is determined by the intrinsic properties of alpha-syn itself and by the cytoplasmic context.     

Electrostatic interaction between retinylidene chromophore and opsin in rhodopsin studied by fluorinated rhodopsin analogues

Photochemical reactions of fluorinated rhodopsin analogues (F-rhodopsins) prepared from 10- or 12-fluorinated retinals (10- or 12-F-retinals) and cattle opsin were investigated by means of low-temperature spectrophotometry. On irradiation with blue light at liquid nitrogen temperature (-191 degrees C), the F-rhodopsins were converted to their respective batho intermediates. On warming, they decomposed to their respective fluororetinals and cattle opsin through lumi and meta intermediates. There was a difference in photochemical behavior between batho-12-F-rhodopsin and batho-10-F-rhodopsin. Upon irradiation with red light at -191 degrees C, batho-12-F-rhodopsin was converted to a mixture of 12-F-rhodopsin and 9-cis-12-F-rhodopsin like that of the natural bathorhodopsin, whereas batho-10-F-rhodopsin was not converted to 9-cis-10-F-rhodopsin but only to 10-F-rhodopsin. This fact suggests that the fluorine substituent at the C10 position (i.e., 10-fluoro) of the retinylidene chromophore may interact with the protein moiety during the process of isomerization of the chromophore or in the state of the batho intermediate. On irradiation with blue light at -191 degrees C, 9-cis-10-F-rhodopsin was converted to another bathochromic intermediate that was different in absorption spectrum from batho-10-F-rhodopsin. 9-cis-10-F-rhodopsin was practically "photoinsensitive" at liquid helium temperature (-265 degrees C), whereas 10-F-rhodopsin was converted to a photo-steady-state mixture of 10-F-rhodopsin and batho-10-F-rhodopsin. The specific interaction between the fluorine atom at the C10 position of the retinylidene chromophore and the opsin was discussed in terms of electrostatic interactions.     

Electrostatic and lipid anchor contributions to the interaction of transducin with membranes: mechanistic implications for activation and translocation

The heterotrimeric G protein transducin is a key component of the vertebrate phototransduction cascade. Transducin is peripherally attached to membranes of the rod outer segment, where it interacts with other proteins at the membrane-cytosol interface. However, upon sustained activation by light, the dissociated G(t)alpha and Gbeta(1)gamma(1) subunits of transducin translocate from the outer segment to other parts of the rod cell. Here we used a computational approach to analyze the interaction strength of transducin and its subunits with acidic lipid bilayers, as well as the range of orientations that they are allowed to occupy on the membrane surface. Our results suggest that the combined constraints of electrostatics and lipid anchors substantially limit the rotational degrees of freedom of the membrane-bound transducin heterotrimer. This may contribute to a faster transducin activation rate by accelerating transducin-rhodopsin complex formation. Notably, the membrane interactions of the dissociated transducin subunits are very different from those of the heterotrimer. As shown previously, Gbeta(1)gamma(1) experiences significant attractive interactions with negatively charged membranes, whereas our new results suggest that G(t)alpha is electrostatically repelled by such membranes. We suggest that this repulsion could facilitate the membrane dissociation and intracellular translocation of G(t)alpha. Moreover, based on similarities in sequence and electrostatic properties, we propose that the properties described for transducin are common to its homologs within the G(i) subfamily. In a broader view, this work exemplifies how the activity-dependent association and dissociation of a G protein can change both the affinity for membranes and the range of allowed orientations, thereby modulating G protein function.