Mesostructure of fibrillar bovine serum albumin gels

The mesostructure of bovine serum albumin (BSA) at low pH was investigated. Rheological measurements were performed to determine the critical percolation concentration (c_p). A decreasing c_p with increasing ionic strength was found. Fibrils with a contour length of about 100–300 nm were found using transmission electron microscopy. The measured conversion of monomers into fibrils was independent of ionic strength (0.20–0.30 M). Dilution of BSA samples showed that the aggregation process is reversible and that there exists a critical concentration for the self-assembly of BSA. We explain the decreasing c_p with increasing ionic strength in terms of an adjusted random contact model.     

Swelling and dissolution of beta-lactoglobulin gels in alkali

It is well documented in the literature that during the dissolution of whey protein gels in alkali, the gels swell to a great extent. However, the relevance of the swelling step in the dissolution process of the protein gel remains unknown. In the present article we present a systematic study on the swelling of beta-lactoglobulin gels at different alkaline pH and ionic strengths. The equilibrium swelling degree at different conditions has been modeled using a simple model developed for polyelectrolyte gels, modified to take into account the ionization of the residues in a protein. The model can describe the swelling behavior of the gels over a wide range of conditions, but it underpredicts the equilibrium swelling under conditions close to those when dissolution is observed. Dissolution is only noticeable above pH 11.5-12 and only for those gels that are swollen over a minimum degree, suggesting the existence of a dissolution threshold.     

Effect of electrostatic interactions on the ultrafiltration behavior of charged bacterial capsular polysaccharides

Charged polysaccharides are used in the food industry, as cosmetics, and as vaccines. The viscosity, thermodynamics, and hydrodynamic properties of these charged polysaccharides are known to be strongly dependent on the solution ionic strength because of both inter‐ and intramolecular electrostatic interactions. The goal of this work was to quantitatively describe the effect of these electrostatic interactions on the ultrafiltration behavior of several charged capsular polysaccharides obtained from Streptococcus pneumoniae and used in the production of Pneumococcus vaccines. Ultrafiltration data were obtained using various Biomax? polyethersulfone membranes with different nominal molecular weight cutoffs. Polysaccharide transmission decreased with decreasing ionic strength primarily because of the expansion of the charged polysaccharide associated with intramolecular electrostatic repulsion. Data were in good agreement with a simple theoretical model based on solute partitioning in porous membranes, with the effective size of the different polysaccharide serotypes evaluated using size exclusion chromatography at the same ionic conditions. These results provide fundamental insights and practical guidelines for exploiting the effects of electrostatic interactions during the ultrafiltration of charged polysaccharides. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1531–1538, 2016     

Robust cross-links in molluscan adhesive gels: testing for contributions from hydrophobic and electrostatic interactions

The cross-linking interactions that provide cohesive strength to molluscan adhesive gels were investigated. Metal-based interactions have been shown to play an important role in the glue of the slug Arion subfuscus (Draparnaud), but other types of interactions may also contribute to the glue's strength and their role has not been investigated. This study shows that treatments that normally disrupt hydrophobic or electrostatic interactions have little to no effect on the slug glue. High salt concentrations and non-ionic detergent do not affect the solubility of the proteins in the glue or the ability of the glue proteins to stiffen gels. In contrast, metal chelation markedly disrupts the gel. Experiments with gel filtration chromatography identify a 40 kDa protein that is a central component of the cross-links in the glue. This 40 kDa protein forms robust macromolecular aggregations that are stable even in the presence of high concentrations of salt, non-ionic detergent, urea or metal chelators. Metal chelation during glue secretion, however, may block some of these cross-links. Such robust, non-specific interactions in an aqueous environment are highly unusual for hydrogels and reflect an intriguing cross-linking mechanism.     

Dimerization, stability and electrostatic properties of porcine beta-lactoglobulin.

The study of homologous proteins belonging to the same family can provide a rationale for important molecular properties such as oligomer formation, folding mechanism and mode of binding. We report here a physico-chemical characterization of porcine beta-lactoglobulin, purified from pooled milk: size-exclusion chromatography, CD and NMR measurements were used to study the aggregation and stability of this protein. In spite of the high sequence identity and homology of porcine beta-lactoglobulin with the widely studied bovine species, the two proteins exhibit very different behaviours. The porcine protein shows a monomer-dimer equilibrium with a pH dependence opposite to that observed for the bovine species. Unfolding experiments revealed the presence of an intermediate that probably has excess alpha helices, as reported for equine species. Modelling studies were performed on bovine, porcine and equine proteins, and, interestingly, electrostatic surface potential calculations led to results consistent with the different dimer interface found for porcine beta-lactoglobulin in the crystal structure. Interaction studies revealed that porcine beta-lactoglobulin is unable to bind fatty acids at any pH, thus questioning the main functional role proposed for lactoglobulins as fatty acid transporters or solubilizers.     

Effect of temperature and concentration on rheological behavior of xanthan-carob mixed gels

The gelation and melting behavior of 1∶1, 1∶3 xanthan-carob mixed gels were evaluated at isothermal and non-isothermal states, as a function of total polymer concentrations of 0.1, 0.5 and 1%. A thermal hysteresis was observed between gelation and melting. The higher the polymer concentration, the higher the melting temperature. The gelation points were determined by three criteria. Depending on the criterion used the gelation temperature was different (52 to 70°C). Pseudoequilibrium modulus and elastic active network chain (EANC) concentration were calculated from the plateau modulus in the frequency spectrum. Temperature dependence of the monomeric friction coefficient was estimated from the relaxation time and EANC. Time-temperature superposition theory was not applicable due to dramatic phase transitions occurring during the gelation of X/C mixture.     

Effect of pH and acrylamide concentration on the separation of lipopolysaccharides in polyacrylamide gels

The technique of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the O-antigen of three strains ofPseudomonas aeruginosa, two strains, ofSalmonella typhimurium, and one strain ofEscherichia coli. No significant difference in separation and migration rate of sample was seen at the various acrylamide gel concentrations used. However, samples electrophoresed through acrylamide running gels at pH 6.8 migrated faster and the resolution of the high-molecular-weight O-antigen bands was greater than of the samples separated in gels at pH 8.8. On the basis of our observations, we could conclude that separation of the heterogeneous O-antigen in SDS-PAGE is probably due to differences in their charge densities and their molecular sizes. Also, pH 6.8 resolving gels are especially useful in the separation of high-molecular-weight O-antigen for epitope mapping by reaction with monoclonal antibodies in Western immunoblotting.     

Circular dichroism study on the early folding events of beta-lactoglobulin entrapped in wet silica gels

beta-Lactoglobulin is a predominantly beta-sheet protein that folds by forming excess alpha-helices within milliseconds. In this study, the refolding of beta-lactoglobulin was dramatically decelerated by entrapping in wet nanoporous silica gel matrices, and monitored on a time scale of minutes or hours by far-UV circular dichroism spectroscopy. Analysis of kinetics and transient spectra allowed to define the sequence of folding events that consist of alpha-helical formation, beta-sheet core formation, and alpha-to-beta transition. The results suggest that the initially formed alpha-helices, presumably including the native alpha-helix, help to guide the formation of the adjacent beta-sheet core.     

Molecular differences in the formation and structure of fine-stranded and particulate beta-lactoglobulin gels

In order to reveal at a molecular level differences between fine-stranded and particulate gels, we present an Fourier transform infrared spectroscopic study of the thermal behavior of beta-lactoglobulin (beta-lg) in salt-free D(2)O solutions and low ionic strength at different pDs. Differences are found in the denaturation mechanism, in the unfolded state of the protein, in the aggregate formation, and in the strength of the intermolecular interactions. For fine-stranded gels (pD 2.8 and 7.8), heating induces the dissociation of the dimers into monomers. The protein undergoes extensive structural modifications before aggregation begins. Aggregation is characterized by the appearance of a new band attributed to intermolecular beta-sheets which is located in the 1613-1619 cm(-1) range. For particulate gels (pD 4.4 and 5.4), the protein structure is almost preserved up to 75-80 degrees C with no splitting of the dimers. The band characteristic of aggregation originates from the component initially located at 1623 cm(-1), suggesting that at the beginning of aggregation, globular beta-lg in the dimeric form associate to constitute oligomers with higher molecular mass. Aggregation may result in the association of globular slightly denatured dimers, leading to the formation of spherical particles rather than linear strands. The aggregation band is always located in the 1620-1623 cm(-1) range for particulate gels showing that hydrogen bonds are weaker for these aggregates than for fine-stranded ones. This has been related to a more extensive protein unfolding for fine-stranded gels that allows a closer alignment of the polypeptide chains, and then to the formation of much stronger hydrogen bonds. Small differences are also found in protein organization and in intermolecular hydrogen bond strength vs pD within the same type of gel. Protein conformation and protein-protein interactions in the gel state may be responsible of the specific macroscopic properties of each gel network. A coarse representation of the different modes of gelation is described.     

The pH threshold in the dissolution of beta-lactoglobulin gels and aggregates in alkali

The existence of a practical minimum pH for the dissolution of heat-induced whey gels in alkaline solutions has been studied using beta-lactoglobulin (betaLg) as a model protein. A sharp transition in solubility was observed between pH 11 and 12; this transition shifts to higher pHs for gels formed at higher temperatures and for longer gelling times. The breakdown reactions of heat-induced aggregates in alkali were monitored with size exclusion chromatography. The destruction of large aggregates was faster at higher pH and also showed a transition between pH 11 and 12. Using tryptophan fluorescence and near- and far-UV circular dichroism, this transition was assigned to the base-induced denaturation observed in solutions of aggregates (pK 11.53). It is suggested that the high protein repulsion caused by the large number of charges at pH > 11.5 drives the unfolding of the protein and the disruption of the intermolecular noncovalent bonds. Concentrated urea and GuHCl were found to be less effective than a pH 12 solution in destroying large aggregates. Aggregates formed for a long time (80 degrees C for 24 h) contained a larger number of intermolecular disulfide bonds that hinder the dissolution process. Gels formed at low temperatures (65 degrees C for 60 min), with fewer intermolecular noncovalent bonds, showed a similar solubility-pH profile to that observed for the base-induced denaturation of unheated beta-lactoglobulin (betaLg) (pK 10.63).     

Effect of liposome charge and PEG polymer layer thickness on cell-liposome electrostatic interactions

Targeted drug delivery requires binding to (and subsequent uptake by) the carrier and target cell. In this paper, we calculate the work required to bring into contact liposomal carriers and cells as a function of the liposome and cell electrostatic characteristics. We find that cell-liposome adhesion is sensitive to the cell type and optimized at a cell to liposome charge ratio which depends on the degree of cell charge regulation. As a result, uptake (which is dependent on the occurrence of binding) is also optimized. Incorporation of a (poly)ethylene glycol (PEG) layer enhances liposome adhesion in cases where the cell-liposome interactions are repulsive, and suppresses adhesion in systems where the interactions are attractive. Our results, which are in agreement with experimental observations, show that electrostatic interactions may be designed to enable targeted drug delivery by liposomes to a specific cell population.     

Peptides are building blocks of heat-induced fibrillar protein aggregates of beta-lactoglobulin formed at pH 2

The proteinaceous material present in beta-lactoglobulin fibrils formed after heating (20 h at 85 degrees C) at pH 2 was identified during this study. Fibrils were separated from the nonaggregated material, and the fibrils were dissociated using 8 M guanidine chloride and 0.1 M 1,4-dithiothreitol (pH 8). Characterization of the different fractions was performed using thioflavin T fluorescence, high-performance size-exclusion chromatography, reversed-phase HPLC, and mass spectrometry (MALDI-TOF). Beta-lactoglobulin was found to be hydrolyzed into peptides with molecular masses between 2000 and 8000 Da, and the fibrils were composed of a part of these peptides and not intact beta-lactoglobulin. The majority of the peptides (both aggregated and nonaggregated) were a result from cleavage of the peptide bonds before or after aspartic acid residues. Explanations for the presence of certain peptide fragments in the fibrils are the hydrophobicity, low charge, charge distribution, and capacity to form beta-sheets.     

Effect of liposome charge and PEG polymer layer thickness on cell-liposome electrostatic interactions

Targeted drug delivery requires binding to (and subsequent uptake by) the carrier and target cell. In this paper, we calculate the work required to bring into contact liposomal carriers and cells as a function of the liposome and cell electrostatic characteristics. We find that cell-liposome adhesion is sensitive to the cell type and optimized at a cell to liposome charge ratio which depends on the degree of cell charge regulation. As a result, uptake (which is dependent on the occurrence of binding) is also optimized. Incorporation of a (poly)ethylene glycol (PEG) layer enhances liposome adhesion in cases where the cell-liposome interactions are repulsive, and suppresses adhesion in systems where the interactions are attractive. Our results, which are in agreement with experimental observations, show that electrostatic interactions may be designed to enable targeted drug delivery by liposomes to a specific cell population.     

Effect of surface electrostatic interactions on the stability and folding of formate dehydrogenase from Candida methylica

NAD⁺-dependent formate dehydrogenase (FDH-EC 1.2.1.2) is an important enzyme to regenerate valuable NADH required by NAD⁺-dependent oxidoreductases in enzyme catalysis. The limitation in the thermostability of FDH enzyme is a crucial problem for development of biotechnological and industrial processes, despite of its advantages. In this study, to investigate the contribution of surface electrostatic interaction to the thermostability of FDH from Candida methylica (cmFDH) N187E, H13E, Q105R, N300E, N147R N300E/N147R, N187E/Q105R, N187E/N147R,Y160R, Y302R, Y160E and Y302E mutants were designed using a homology model of cmFDH based on Candida boidinii (cb) by considering electrostatic interactions on the protein surface. The effects of site-specific engineering on the stability of this molecule was analyzed according to minimal model of folding and assembly reaction and deduced equilibrium properties of the native system with respect to its thermal and denaturant sensitivities. It was observed that mutations did not change the unfolding pattern of native cmFDH and increased numbers of electrostatic interactions can cause either stabilizing or destabilizing effect on the thermostability of this protein. The thermodynamic and kinetic results suggested that except relatively improved mutants, three out of the nine single mutations increased the melting temperature of cmFDH enzyme.     

Effect of pH on the ternary solution behavior of beta-lactoglobulin

Ternary phase diagrams (TPDs) were constructed for aqueous beta-lactoglobulin solutions containing ethanol and (NH4)2SO4 at pHs of 7, 5, and 3 for temperatures between 20 and 70 degrees C. The addition of (NH4)2SO4 generally led to the production of a reversible precipitate, a transformation that was not strongly influenced by temperature or pH. In contrast, at pH 7 and 20 degrees C, ethanol concentrations >12% led to the formation of a molten-globule structure, which gelled at protein concentrations >10%. Destabilization of beta-lactoglobulin structure occurred at lower ethanol concentrations as temperature was increased, until at 70 degrees C, all solutions that were previously liquid at room temperature had transformed into a gel. At pH 5.0, near beta-lactoglobulin's isoelectric point, demixing dominated, leading to the creation of either irreversible precipitates or a paste-like microgel. Elevated temperatures caused the previously liquid morphology to transform into either a reversible aggregate or microgel. Solution behavior at pH 3 had characteristics of what was observed at pHs 7 and 5. At moderated protein and ethanol concentrations, a paste-like microgel was observed, whereas at higher ethanol concentrations, beta-lactoglobulin formed a gel. This work demonstrates how small changes in protein structure at the molecular level can have a dramatic effect on macroscopic morphology.     

Influence of the solvent structure on the electrostatic interactions in proteins

The proper estimation of the influence of the many-body dynamic solvent microstructure on a pairwise electrostatic interaction (PEI) at the protein-solvent interface is very important for solving many biophysical problems. In this work, the PEI energy was calculated for a system that models the interface between a protein and an aqueous solvent. The concept of nonlocal electrostatics for interfacial electrochemical systems was used to evaluate the contribution of a solvent orientational polarization, correlated by the network of hydrogen bonds, into the PEI energy in proteins. The analytical expression for this energy was obtained in the form of Coulomb's law with an effective distance-dependent dielectric function. The asymptotic and numerical analysis carried out for this function revealed several features of dielectric heterogeneity at the protein-solvent interface. For charges located in close proximity to this interface, the values of the dielectric function for the short-distance electrostatic interactions were found to be remarkably smaller than those determined by the classical model, in which the solvent was considered as the uniform dielectric medium of high dielectric constant. Our results have shown that taking into consideration the dynamic solvent microstructure remarkably increases the value of the PEI energy at the protein-solvent interface.     

Fibrillar beta-lactoglobulin gels: Part 3. Dynamic mechanical characterization of solvent-induced systems

Oscillatory shear rheometry has been used to study the gelation of beta-lactoglobulin at ambient in 50% v/v trifluoroethanol (TFE)/pH 7 aqueous buffer and in 50% v/v ethanol (EtOH)/water at pH 2. In contrast to what was found on heating aqueous solutions at pH 2 (Part 2 of this series), a more expected "chemical gelation"-like profile was found with modulus components G' and G' ' crossing over as the gels formed and then with G' ' passing through a maximum. In addition, for the EtOH system, there was a significant modulus increase at long time, suggestive of a more complex two-step aggregation scheme. Modulus-concentration relationships were obtained for both systems by extrapolating cure data to infinite time. For the TFE gels, this data was accurately described by classical branching theory, although it could also be approximated by a constant power--law relationship. Only the latter described the modulus--concentration data for the gels in ethanol, but there were problems here of greater frequency dependence of the modulus values and much less certain extrapolation. Gel times for the TFE systems showed higher power laws in the concentration than could be explained by the branching theory in its simplest form being similar, in this respect, to the heat-set systems at pH 2. Such power laws were harder to establish for the EtOH gels as for these there was evidence of gel time divergence close to a critical concentration. Reduced G'/G'inf versus t/tgel data were difficult to interpret for the gels in ethanol, but for the TFE system they were consistent with previous results for the heat-set gels and approximated master curve superposition. The frequency and temperature dependences of the final gel moduli were also studied. In general, the networks induced by alcohols appeared more flexible than those obtained by heating.     

On the calculation of electrostatic interactions in proteins

In this paper we present a classical treatment of electrostatic interactions in proteins. The protein is treated as a region of low dielectric constant with spherical charges embedded within it, surrounded by an aqueous solvent of high dielectric constant, which may contain a simple electrolyte. The complete analysis includes the effects of solvent screening, polarization forces, and self energies, which are related to solvation energies. Formulae, and sample calculations of forces and energies, are given for the special case of a spherical protein. Our analysis and model calculations point out that any consistent treatment of electrostatic interactions in proteins should account for the following. Solvent polarization is an important factor in the calculation of pairwise electrostatic interactions. Solvent polarization substantially affects both electrostatic energies and forces acting upon charges. No simple expression for the effective dielectric constant, Deff, can generally be valid, since Deff is a sensitive function of position. Solvent screening of pairwise interactions involving dipolar groups is less effective than the screening of charges. In fact for many interactions involving dipoles, solvent screening can be essentially ignored. The self energy of charges makes a large contribution to the total electrostatic energy of a protein. This must be compensated by specific interactions with other groups in the protein. Strategies for applying our analysis to proteins whose structures are known are discussed.