Three-Dimensional Structural Stability and Local Electrostatic Potential at Point Mutations in Spike Protein of SARS-CoV-2 Coronavirus.

The contagiousness of SARS-CoV-2 ß-coronavirus is determined by the virus-receptor electrostatic association of its positively charged spike (S) protein with the negatively charged angiotensin converting enzyme-2 (ACE2 receptor) of the epithelial cells. If some mutations occur, the electrostatic potential on the surface of the receptor-binding domain (RBD) could be altered, and the S-ACE2 association could become stronger or weaker. The aim of the current research is to investigate whether point mutations can noticeably alter the electrostatic potential on the RBD and the 3D stability of the S1-subunit of the S-protein. For this purpose, 15 mutants with different hydrophilicity and electric charge (positive, negative, or uncharged) of the substituted and substituting amino acid residues, located on the RBD at the S1-ACE2 interface, are selected, and the 3D structure of the S1-subunit is reconstructed on the base of the crystallographic structure of the S-protein of the wild-type strain and the amino acid sequence of the unfolded polypeptide chain of the mutants. Then, the Gibbs free energy of folding, isoelectric point, and pH-dependent surface electrostatic potential of the S1-subunit are computed using programs for protein electrostatics. The results show alterations in the local electrostatic potential in the vicinity of the mutant amino acid residue, which can influence the S-ACE2 association. This approach allows prediction of the relative infectivity, transmissibility, and contagiousness (at equal social immune status) of new SARS-CoV-2 mutants by reconstruction of the 3D structure of the S1-subunit and calculation of the surface electrostatic potential.

Omicron Coronavirus: pH-Dependent Electrostatic Potential and Energy of Association of Spike Protein to ACE2 Receptor.

The association of the S-protein of the SARS-CoV-2 beta coronavirus to ACE2 receptors of the human epithelial cells determines its contagiousness and pathogenicity. We computed the pH-dependent electric potential on the surface of the interacting globular proteins and pH-dependent Gibbs free energy at the association of the wild-type strain and the omicron variant. The calculated isoelectric points of the ACE2 receptor (pI 5.4) and the S-protein in trimeric form (pI 7.3, wild type), (pI 7.8, omicron variant), experimentally verified by isoelectric focusing, show that at pH 6-7, the S1-ACE2 association is conditioned by electrostatic attraction of the oppositely charged receptor and viral protein. The comparison of the local electrostatic potentials of the omicron variant and the wild-type strain shows that the point mutations alter the electrostatic potential in a relatively small area on the surface of the receptor-binding domain (RBD) of the S1 subunit. The appearance of seven charge-changing point mutations in RBD (equivalent to three additional positive charges) leads to a stronger S1-ACE2 association at pH 5.5 (typical for the respiratory tract) and a weaker one at pH 7.4 (characteristic of the blood plasma); this reveals the reason for the higher contagiousness but lower pathogenicity of the omicron variant in comparison to the wild-type strain.

Composite Hydrogels with Included Solid-State Nanoparticles Bearing Anticancer Chemotherapeutics.

Hydrogels have many useful physicochemical properties which, in combination with their biocompatibility, suggest their application as a drug delivery system for the local and prorogated release of drugs. However, their drug-absorption capacity is limited because of the gel net's poor adsorption of hydrophilic molecules and in particular, hydrophobic molecules. The absorption capacity of hydrogels can be increased with the incorporation of nanoparticles due to their huge surface area. In this review, composite hydrogels (physical, covalent and injectable) with included hydrophobic and hydrophilic nanoparticles are considered as suitable for use as carriers of anticancer chemotherapeutics. The main focus is given to the surface properties of the nanoparticles (hydrophilicity/hydrophobicity and surface electric charge) formed from metal and dielectric substances: metals (gold, silver), metal-oxides (iron, aluminum, titanium, zirconium), silicates (quartz) and carbon (graphene). The physicochemical properties of the nanoparticles are emphasized in order to assist researchers in choosing appropriate nanoparticles for the adsorption of drugs with hydrophilic and hydrophobic organic molecules.

Protein-Mineral Composite Particles with Logarithmic Dependence of Anticancer Cytotoxicity on Concentration of Montmorillonite Nanoplates with Adsorbed Cytochrome c.

Montmorillonite (MM) colloid nanoplates have high adsorption capacity due to their large size/thickness ratio, which allows them to be used as carriers for drug delivery. Upon adsorption of the mitochondrial protein cytochrome c (cytC) onto MM plates, the composite cytC-MM particles acquire anticancer properties because of the ability of cancer cells to phagocytize submicron particles (in contrast to the normal cells). In this way, exogenous cytC can be introduced into tumor cells, thereby triggering apoptosis-an irreversible cascade of biochemical reactions leading to cell death. In the present study, we investigated the physicochemical properties of cytC-MM particles as a function of the cytC concentration in the suspension, namely, the electrophoretic mobility, the mass increment of MM monoplates upon cytC adsorption, the ratio of the adsorbed to the free cytC in the bulk, the protein density on the MM's surface, the number of cytC globules adsorbed on an MM monoplate, the concentration of cytC-MM composite particles in the suspension, and the dependence of cytotoxicity on the cytC-MM particle concentration. For this purpose, we used microelectrophoresis, static and electric light scattering, and a colon cancer cell culture to test the cytotoxic effects of the cytC-MM suspensions. The results show that the cytotoxicity depends linearly on the logarithm of the particle concentration in the cytC-MM suspension reaching 97%.

Spontaneous and Electrically Induced Anisotropy of Composite Agarose Gels.

Agarose gels containing and not bacteriorhodopsin purple membranes (incorporated before gelling) manifest spontaneous optical anisotropy. The dependencies of the anisotropy on the agarose concentration and time have been studied. The rise in the anisotropy is explained by the predominant orientation of the agarose fibers during the gelling and subsequent deformation of the gel net. In the electric field, additional optical anisotropy rises, which is caused by the orientation of the membranes. A procedure has been developed to separate electrically induced and spontaneous anisotropy in composite gels. The isoelectric points and surface electric potential of bacteriorhodopsin trimer and purple membranes are calculated by the method of protein electrostatics to explain their electric asymmetry, which leads to perpendicular orientation in the direct electric field and longitudinal in the kilohertz sinusoidal field. The results allow for an increase in the separation capability of composite gels of electrophoresis for macromolecules with different sizes by applying an appropriate electric field to modulate the effective pore size.

Electrooptical determination of the isoelectric point of globular proteins: Cytochrome c adsorbed on montmorillonite nanoplates.

The purpose is to study the capability of the electric light scattering to determine the point of zero charge of native protein macromolecules adsorbed on colloid particles at low ionic strength and without using buffers. The chosen protein and particles are cytochrome c (cytC, globular haemoproteid) and montmorillonite (MM, negatively charged plate-like crystal). The pH-dependence of the electric polarizability γ(pH) in the range pH 6-11 shows minimum which coincides with the isoelectric point determined by measuring the electrophoretic mobility µ(pH) of the cytC-MM particles.

Isoelectric point of free and adsorbed cytochrome c determined by various methods.

The purpose is to determine the isoelectric point (IEP) pIµ of cytochrome c (cytC, a globular haemoproteid) adsorbed on montmorillonite (MM, plate-like colloid particles) by microelectrophoresis and to compare the pIµ value with pIf9.44 measured by isoelectric focusing in gel with covalently linked ampholytes, and with pInz10.0-10.6 of free cytC globule calculated by three programs for pH-dependent net charge nz using the crystallographic structure of cytC. The pH-dependence of the electrophoretic mobility µ(pH) in the range pH 6-11 shows out that IEP of cytC-MM particles appears at pH 9.35. The near courses of µ(pH) and nz(pH) reveal that the pH-independent negative charge of the MM substrate is hydrodynamically shielded by the adsorbed protein globules. The nearness of IEP and pIf allows attributing IEP value of cytC-MM particles to the isoelectric point pIµ of cytC. A short survey for pI of cytC reported in the literature since 1941 shows out that pI is dispersed in the range pH 9.0-10.65 although cytC is used now as pIf marker with well known IEP; the reason for that and the imperfections of the employed methods are discussed.

Adsorption of cytochrome c on montmorillonite nanoplates: protein concentration dependence.

Cytochrome c [cytC] is a mitochondrial hemoprotein functioning as electron carrier in the respiratory chain of the biological cells. Being adsorbed on colloid particles cytC can be introduced in the cells by phagocytoses. In the present work we study the adsorption of cytC on montmorillonite (MM) particles combining the electro-optic and electrophoretic techniques. MM particles were chosen as nanoplates having negative pH-independent charge and high ratio surface/mass. The measurements were done at pH 6.5 where cytC globule is positively charged. The main employed method is the electric light scattering based on orientation of colloid particles in sinusoidal electric field. Interfacial electric polarizability was obtained from the degree of orientation at steady-state and the particle size - from the relaxation time after the field switching off. Microelectrophoresis was used to monitor the alteration of the surface charge at protein adsorption. The cytC-concentration dependence of the polarizability and the mobility shows out that the total (net) charge of cytC-MM complex turns its sign from negative to positive, the isoelectric point appears at 5:3 mg/mg (0.135 mol/kg) cytC/MM and saturated protein adsorption is reached at additional twofold increasing of cytC/MM ratio. The suspension is stable at low and high protein concentrations, at intermediate ones aggregation arises.

Adsorption of carboxymethyl cellulose on alumina particles.

The polyelectrolyte adsorption on colloid particles is often used for stabilization or flocculation of water suspensions. The aim of this work is to study the adsorption of carboxymethyl cellulose (CMC) on alumina (γ-Al2O3) colloid particles. The particles and polymer are chosen because of the capability of the metal-oxide ampholyte surface and the weak polyelectrolytes to alter their charge by pH. The measurements are done at pH 6.0 where the CMC carboxylic gropes are almost fully dissociated and the alumina surface is positively charged. The high linear charge density of the polyelectrolyte chain provides Na(+) counterions condensation on the COO(-) groups. The main employed method is the electric light scattering based on particle orientation in sinusoidal electric field. The electric polarizability and the relaxation time after field switching off (both depending on the particle charge and size) are used as criteria for polymer adsorption and particle aggregation. Micro-electrophoresis is applied as additional techniques indicating the sign and density of the surface charge. The results obtained give the conditions (time dependence, particle and polymer concentrations) where the CMC adsorption is complete and the suspension is stable.

Polymer concentration dependence of kilohertz electric polarizability of alumina colloid particles with adsorbed carboxymethyl cellulose.

Polyelectrolytes are soluble polymers composed of units having charged groups. Because of the high charge density, some of the counterions are adsorbed electrostatically (ion condensation) on the polyelectrolyte chain. It was shown that in direct electric field the condensed counterions and the chain move together as one whole but it is assumed that they are mobile in alternating field and participate in the polarization. Experimental evidence is obtained by electro-optical investigations of polyelectrolytes adsorbed on colloid particles-the observed low-frequency shift of the polarizability relaxation has been interpreted as condensed counterions' mobility. The present investigation aims to verify the reports for the condensed counterions' mobility in sinusoidal electric field. By means of electric light scattering we investigated a water suspension of γ-alumina particles with adsorbed carboxymethyl cellulose. Instead of the previously used frequency approach (dispersion dependence at saturated adsorption of the polyelectrolyte) we applied an amplitude approach-determination of the polarizability at frequency 1 kHz and increasing polyelectrolyte concentration (from zero to full adsorption saturation). The results indicate the absence of polarization owing to the condensed counterions. The main evidence was obtained by comparison of the concentration dependences of the polarizability (depending on all mobile counterions) and the electrophoretic mobility (determined only by the diffuse counterions). We concluded that the condensed counterions are immobile in sinusoidal field with intensity up to 0.5  kV cm (- 1) and frequency of 1 kHz and higher.

Electric polarizability changes during E. coli culture growth.

The electric polarizability of bacteria has two main components: surface-charge dependent (SChD) and Maxwell-Wagner (MW). It has been reported that the low frequency SChD component of Escherichia coli K12 still arise in the frequency range 20kHz - 2MHz, together with the high-frequency MW one. All the previous experiments were carried out with bacterial cultures of E. coli K12 in the stationary phase. In the present work we study electric polarizability during culture growth with the aim of finding out how it is influenced by the physiological state of the cells. The electro-optical method of electric turbidimetry is used, which is based on the change in the optical density as a result of orientation of bacterial cells under the action of an applied electric field. Our results show that until the cell concentration increases exponentially, the polarizability and the cell size change synchronously, so that the polarizability is approximately a quadratic function of the average bacterial length. We explain this with dominance of the SChD component. However, that after the polarizability deceases twofold at insignificant length oscillations and the power of the function decreases to 1.5. The last result is interpreted as an increase in the MW component.

Influence of the medium electrolyte concentration on the electric polarizability of bacteria Escherichia coli in presence of ethanol.

The electric polarizability is an important parameter of bacteria, giving information about the electric properties of the cells. In our previous works [A.M. Zhivkov, A.Y. Gyurova, Colloids Surf. B: Biointerfaces 66 (2008) 201; A.Y. Gyurova, A.M. Zhivkov, Biophys. Chem., 139 (2009) 8; A.M. Zhivkov, A.Y. Gyurova, J. Phys. Chem. B, 113 (2009) 8375] we have applied an experimental approach to distinguish the contribution of the components of the two types of interface electric polarizability-surface charge dependent (ChD) and Maxwell-Wagner (MW) polarizability. It is based on electro-optical study of the separate influence of the outer and inner medium electrolyte concentration, which changes the external ChD and internal MW components of polarizability; the last effect is reached by the membrane permeability increase in low ethanol concentration. In the present work we investigate the behavior of electric polarizability of Escherichia coli K12 at increasing the outer KCl concentration in presence of 10 vol.% ethanol in order to check if the polarizability components change independently from one another. The conclusion is that the outer electrolyte concentration influence indirectly the internal MW component by the trans-membrane concentration gradient, but the polarizability components themselves change independently.

Influence of cytoplasm electrolyte concentration on Maxwell-Wagner polarizability of bacteria E. coli.

The electric polarizability of bacteria is considered in the literature to have a surface charge dependent (ChD) and a Maxwell-Wagner (MW) mechanism. We distinguish experimentally both the types of interface polarizability by the frequency of the electric field and the medium electrolyte concentration. It was shown in a previous work ( Zhivkov , A. M. ; Gyurova , A. Y. Colloids Surf., B 2008 , 66 , 201. ) that the ChD component is shown up on the outer bacteria surface even at megahertz frequencies. The MW polarizability is studied in the present work in the range from 20 kHz to 20 MHz by change in the inner (cytoplasm) electrolyte concentration. The ion transport through the cytoplasmic membrane of alive and fixed by formaldehyde E. coli K12 is accelerated by adding of ethanol in low concentration. The frequency dependence and the kinetics of the electric polarizability and the size of the bacteria are investigated by conservative electric dichroism, based on the alteration of the optical density at orientation of the cells in electric field. The conclusion is that the internal MW component has the main contribution to the change in the total bacteria polarizability, as well as the external MW and the internal ChD components are not shown up.

Influence of ethanol on the high frequency electric polarizability of E. coli.

The interface electric polarizability of bacteria (charge dependent (ChD) and Maxwell-Wagner (MW) polarizabilities) gives information about their electric charge, determined by the structure and functional state. It is well known that the polarizability could be changed significantly by adding some substances to the suspension, and can be measured using an electro-optical (EO) method. There are some literature data, according to which the adding of ethanol decreases the electric polarizability of the cells. However the reason for the change in this parameter is not clear, as well as which component (ChD and/or MW) of polarizability has the main contribution. Generally the present work shows that the effect of ethanol is connected to the change of the internal (cytoplasm) MW polarizability and is mainly caused by increasing the cell membrane permeability. This results in an ionic flow through the membrane, which velocity and direction depends on the relative values of the inner (cytoplasm) and the outer medium ionic strength.

Dependence of depletion layer thickness on polymer concentration determined by Vincent's pragmatic and Donath's electrophoretic theories.

On the interface of a solid surface and a solution of nonadsorbing polymer there exists a depletion layer (DL), where the concentration of polymer segments is lower. Donath's electrophoretic theory, based on the decreased viscosity in the DL region, allows computing DL thickness from the relative (with and without polymer) electrophoretic mobility, the bulk viscosity, and the ionic strength. The aim of this work is to check experimentally Donath's nonlinear electrophoretic (NLE) theory under the most favorable conditions--liposomes in solutions of low-molecular poly(ethylene glycol) (PEG). In order to determine DL thickness, the dependence of mobility on viscosity is chosen instead on ionic strength. The value obtained from NLE theory is compared with the DL thickness calculated by Vincent's pragmatic theory. The conformation-statistical parameters are calculated on the base of viscosimetric measurements of PEG solution. The results indicate a few shortcomings of NLE theory. The main one is that DL thickness does not depend on polymer concentration, a fact that is in discrepancy with the prediction of Vincent's theory. The conclusion is that NLE theory describes well the experimental dependence of the relative mobility on the bulk viscosity, but it is inapplicable to quantitative determination of DL thickness.

Change of purple membranes geometry induced by protein adsorption.

Earlier it was an orthodoxy that purple membranes (PMs) in aqueous medium are shaped as flat hard disks. In a few newer articles it has been shown that PMs are bent and their curvature varies with surface charge density. The purpose of this work is to answer which is the dominant factor for PM bending--structural or electrostatic forces. Two positively charged proteins are used: phytohemagglutinin (PhHA) and protamine. The electrophoretic mobility and electric polarizability of PMs are measured by microelectrophoresis and electric dichroism. The results show that both proteins reduce the mobility because they are adsorbed on PM surface. However, their influence on the electric polarizability is in the opposite direction--protamine reduces it (trivial effect) while PhHA increases the polarizability (non-trivial effect). The last result is explained by a straightening the initially bent PM because of specific bonding of PhHA to asymmetrically disposed glycolipids of PM in contrast to the electrostatic adsorption of protamine. It has been concluded that PMs in water medium are bent in the same manner as in in vivo--the intracellular surface with a higher negative charge is concave. The results indicate that electrostatic forces play a significant role in PM curvature but the shape of structural elements is the main factor determining the geometry of PM.