Net Repulsive Interactions Research Articles

Use of Debye–Hückel–Henry charge measurements in early antibody development elucidates effects of non-specific association

The diffusion interaction parameter (kD) has been demonstrated to be a high-throughput technique for characterizing interactions between proteins in solution. kD reflects both attractive and repulsive interactions, including long-ranged electrostatic repulsions. Here, we plot the mutual diffusion coefficient (Dm) as a function of the experimentally determined Debye–Hückel–Henry surface charge (ZDHH) for seven human monoclonal antibodies (mAbs) in 15 mM histidine at pH 6. We find that graphs of Dm versus ZDHH intersect at ZDHH, ~ 2.6, independent of protein concentration. The same data plotted as kD versus ZDHH show a transition from net attractive to net repulsive interactions in the same region of the ZDHH intersection point. These data suggest that there is a minimum surface charge necessary on these mAbs needed to overcome attractive interactions.

Steric Repulsion Forces Contributed by PEGylation of Interleukin-1 Receptor Antagonist Reduce Gelation and Aggregation at the Silicone Oil-Water Interface

Silicone oil, used as a lubricating coating in pharmaceutical containers, has been implicated as a cause of therapeutic protein aggregation. After adsorbing to silicone oil-water interfaces, proteins may form interfacial gels, which can be transported into solution as insoluble aggregates if the interfaces are perturbed. Mechanical interfacial perturbation of both monomeric recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and PEGylated rhIL-1ra (PEG rhIL-1ra) in siliconized syringes resulted in losses of soluble monomeric protein. However, the loss of rhIL-1ra was twice that for PEG rhIL-1ra; even though in solution, PEG rhIL-1ra had a lower ΔGunf and exhibited a more perturbed tertiary structure at the interface. Net protein-protein interactions in solution for rhIL-1ra were attractive but increased steric repulsion because of PEGylation led to net repulsive interactions for PEG rhIL-1ra. Attractive interactions for rhIL-1ra were associated with increases in intermolecular β-sheet content at the interface, whereas no intermolecular β-sheet structures were observed for adsorbed PEG rhIL-1ra. rhIL-1ra formed interfacial gels that were 5 times stronger than those formed by PEG rhIL-1ra. Thus, the steric repulsion contributed by the PEGylation resulted in decreased interfacial gelation and in the reduction of aggregation, in spite of the destabilizing effects of PEGylation on the protein’s conformational stability.

Effects of sleep loss on the cortical microstructure

Silicone oil, used as a lubricating coating in pharmaceutical containers, has been implicated as a cause of therapeutic protein aggregation. After adsorbing to silicone oil-water interfaces, proteins may form interfacial gels, which can be transported into solution as insoluble aggregates if the interfaces are perturbed. Mechanical interfacial perturbation of both monomeric recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and PEGylated rhIL-1ra (PEG rhIL-1ra) in siliconized syringes resulted in losses of soluble monomeric protein. However, the loss of rhIL-1ra was twice that for PEG rhIL-1ra; even though in solution, PEG rhIL-1ra had a lower ΔGunf and exhibited a more perturbed tertiary structure at the interface. Net protein-protein interactions in solution for rhIL-1ra were attractive but increased steric repulsion because of PEGylation led to net repulsive interactions for PEG rhIL-1ra. Attractive interactions for rhIL-1ra were associated with increases in intermolecular β-sheet content at the interface, whereas no intermolecular β-sheet structures were observed for adsorbed PEG rhIL-1ra. rhIL-1ra formed interfacial gels that were 5 times stronger than those formed by PEG rhIL-1ra. Thus, the steric repulsion contributed by the PEGylation resulted in decreased interfacial gelation and in the reduction of aggregation, in spite of the destabilizing effects of PEGylation on the protein’s conformational stability.

Characterization of the NISTmAb Reference Material using small-angle scattering and molecular simulation : Part II: Concentrated protein solutions.

Protein-protein interactions in monoclonal antibody solutions are important for the stability of a therapeutic drug and directly influence viscosity in concentrated protein solutions. This study describes the use of small-angle scattering to estimate protein-protein interactions at high concentrations of the IgG1 NISTmAb reference material and validate colloidal models for interacting molecules. In particular, we studied the colloidal stability of the NISTmAb at high protein concentrations and analyzed protein-protein interactions upon adding sodium chloride and its effect on viscosity. Isotropic colloidal models for interacting molecules were combined with an ensemble of atomistic structures from molecular simulation to account for the flexibility of the NISTmAb in solution. In histidine formulation buffer, net repulsive electrostatic interactions are important for the colloidal stability of the NISTmAb at high concentrations. Addition of sodium chloride increased the viscosity of the NISTmAb and decreased the colloidal stability due to charge screening of the repulsive interactions. The interactions at high concentrations (up to ~ 250mg/mL) were consistent with those from light scattering at low concentrations (below ~ 20mg/mL). However, in the presence of sodium chloride, the screening of charges was less pronounced with increasing protein concentration and the interactions approached those of the repulsive hard-sphere models. Additionally, we studied the NISTmAb under frozen conditions using in situ neutron scattering to analyze the crowded state as proteins are excluded from the water-rich phase. In the frozen samples, where protein concentration can reach hundreds of mg/mL in the protein-rich phase, sodium chloride did not affect the molecular spacing and crowding of the NISTmAb. Graphical Abstract Net repulsive interactions in concentrated NISTmAb solutions assessed by small-angle neutronscattering.

Impact of polymer geometry on the interactions of protein-PEG conjugates

The conjugation of high molecular weight polyethylene glycol (PEG) to an active pharmaceutical ingredient (API) is an attractive strategy for the modification of biophysical and biodistribution properties of the API. Indeed, several therapeutic proteins conjugated to PEG have been safely administered in the clinic. While there have been studies on the configuration of these conjugates in solution, investigations on the impact of PEG geometry on protein-PEG conjugate interactions is limited. In this study, we use dynamic light scattering (DLS), rheology, and small-angle neutron scattering (SANS) to investigate the biophysical solution and interaction behavior of a 50kDa Fab protein attached to either a linear or tetrameric (branched) 40kDa PEG molecule. The hydrodynamic radii, diffusivity, viscosity and pair distance distribution function (PDDF) were obtained for the protein-PEG conjugates in solution. An analysis revealed that interactions between unconjugated proteins were quite attractive, however linear PEG-protein conjugates exhibited net repulsive interactions, similar to that of the unconjugated polymer. Tetramer PEG-protein conjugates on the other hand, exhibited a net weak attractive interaction, indicating a more balanced distribution of repulsive and attractive interaction states. Further analysis of the SANS data using geometric models consistent with the PDDF elucidated the conjugates' equilibrium configuration in solution. Insights gained from measurements and analysis used here can also be useful in predicting how conjugate geometries affect viscosity and aggregation behavior, which are important in determining suitable protein-polymer drug formulations.

Hydrogen Bond Donor/Acceptor Cosolvent-Modified Choline Chloride-Based Deep Eutectic Solvents.

Deep eutectic solvents (DESs) have emerged as nontoxic and inexpensive alternatives not only to the common organic solvents but to the ionic liquids as well. Some of the common and popular, and perhaps the most investigated, DESs are the ones comprising an ammonium salt and an appropriate hydrogen bond (HB) donor in a predetermined mole ratio. The formation of the DES is attributed to the H-bonding interaction(s) present between the salt and the HB donor. Consequently, addition of a predominantly HB donor or a predominantly HB acceptor cosolvent to such DESs may result in intriguing features and properties. We present investigation of two DESs constituted of salt choline chloride along with HB donors urea and glycerol, respectively, in 1:2 mol ratio, named reline and glyceline as the cosolvent of very high HB donating acidity and no HB accepting basicity 2,2,2-trifluoroethanol (TFE) and of very high HB accepting basicity and no HB donating acidity hexamethylphosphoramide (HMPA), respectively, is added. TFE shows up to 0.25 mole fraction miscibility with both reline and glyceline. While up to 0.25 mole fraction HMPA in glyceline results in transparent mixtures, this cosolvent is found to be completely immiscible with reline. From the perspective of the solvatochromic absorbance and fluorescence probes, it is established that the cybotactic region dipolarity within up to 0.25 mole fraction TFE/HMPA-added DES strongly depends on the functionalities present on the solute. Fourier transform infrared absorbance and Raman spectroscopic investigations reveal no major shifts in vibrational transitions as TFE/HMPA is added to the DES; spectral band broadening, albeit small, is observed nonetheless. Excess molar volumes and excess logarithmic viscosities of the mixtures indicate that while TFE may interstitially accommodate itself within H-bonded network of reline, it does appear to form H-bonds with the constituents of the glyceline. Increase in overall net repulsive interactions as HMPA is added to glyceline is suggested by both positive excess molar volumes and excess logarithmic viscosities. The addition of HB donor/acceptor cosolvent appears to disturb the salt-HB donor equilibria within DES via complex interplay of interactions within the system.

Diffusing Colloidal Probes of kT-Scale Biomaterial-Cell Interactions.

In the optimization of applied biomaterials, measurements of their interactions with cell surfaces are important to understand their influence on specific and nonspecific cell surface adhesion, internalization pathways, and toxicity. In this study, a novel approach using dark field video microscopy with combined real-time particle and cell tracking allows the trajectories of biomaterial-coated colloids to be monitored in relation to their distance from cell perimeters. Dynamic and statistical mechanical analyses enable direct measurement of colloid-cell surface association lifetimes and interaction potentials mediated by biomaterials. Our analyses of colloidal transport showed polyethylene glycol (PEG) and bovine serum albumin (BSA) lead to net repulsive interactions with cell surfaces, while dextran and hyaluronic acid (HA) lead to reversible and irreversible association to the cell surface, respectively. Our results demonstrate how diffusing colloidal probes can be used for nonobtrusive, sensitive measurements of biomaterial-cell surface interactions important to therapeutics, diagnostics, and tissue engineering.

Extending surfactant-modified 2:1 clay minerals for the uptake and removal of diclofenac from water

The presence and persistency of pharmaceuticals and personal care products (PPCPs) in the environment attracted great attention recently. Among them, antibiotics and pain-killers accounted for a large quantity. Although many works were devoted to the investigation of their removal in wastewater treatment processes, most of the PPCPs studied were of cationic nature. The net repulsive interactions between anionic PPCPs and negatively charged sorbents make them difficult to be removed in wastewater treatment. In this study, 2:1 clay minerals illite and montmorillonite (MMT) were modified with different amounts of cationic surfactant cetyltrimethylammoium bromide (CTAB). The types and sites of interactions between the surfactant-modified clays and the anionic drug diclofenac (DC) were investigated. The uptake of DC on the modified clays was controlled by the CTAB loading level and its surface configuration on the clays. The adsorption sites of DC were limited to the external surfaces of modified illite due to its non-swelling nature. On the contrary, both the external and interlayer sites were available for the adsorption of DC on modified MMT. A CTAB bilayer formation resulted in significant increase in DC adsorption due to the formation of extensive admicelles. FTIR results showed participation of the benzene ring, NH, and CH2CH3 for the interactions between DC and modified MMT, suggesting that partitioning of DC into the admicelles of CTAB played a significant role in DC uptake. The results could extend the application of surfactant-modified clays for the removal of anionic PPCPs in the wastewater treatment processes.

Interfacial charge rearrangement and intermolecular interactions: Density-functional theory study of free-base porphine adsorbed on Ag(111) and Cu(111).

We employ dispersion-corrected density-functional theory to study the adsorption of tetrapyrrole 2H-porphine (2H-P) at Cu(111) and Ag(111). Various contributions to adsorbate-substrate and adsorbate-adsorbate interactions are systematically extracted to analyze the self-assembly behavior of this basic building block to porphyrin-based metal-organic nanostructures. This analysis reveals a surprising importance of substrate-mediated van der Waals interactions between 2H-P molecules, in contrast to negligible direct dispersive interactions. The resulting net repulsive interactions rationalize the experimentally observed tendency for single molecule adsorption.

Deamidation of Human γS-Crystallin Increases Attractive Protein Interactions: Implications for Cataract.

Deamidation of proteins is one of the most prevalent post-translational modifications found upon aging, and in age-onset diseases. Specific asparagine and glutamine residues are often selectively deamidated during this process. In the human lens, deamidation has been shown to occur in many crystallins, but it is not clear how these deamidated proteins lead to lens opacity or cataract. Here we have modeled in vitro the effect of deamidation of specific asparagine and glutamine residues in human recombinant γS-crystallin (HGS) on the solution properties of the protein. The residues selected for deamidation in vitro are those that are found to be deamidated in aged and cataractous lenses in vivo. Two derivatives were prepared, one with Asn76 and Asn143 deamidated (2N-HGS) and the other with two additional Gln residues (92 and 120) deamidated (2N2Q-HGS). Isoelectric focusing measurements showed the expected lowering of the pI from 6.9 in HGS to ∼6.5 in 2N-HGS and to ∼6.1 in 2N2Q-HGS. However, spectroscopic studies showed no significant change in the secondary and tertiary structures of the deamidated proteins relative to the wild type. The stability of 2N-HGS and 2N2Q-HGS, as measured by guanidinium hydrochloride unfolding, also remained comparable to that of HGS. The main difference was the altered protein-protein interaction among the three proteins. The net repulsive interactions that are characteristic of HGS are diminished in the deamidated derivatives as evidenced by static light scattering measurements of the second virial coefficient, B2 (B2 values for HGS, 2N-HGS, and 2N2Q-HGS of 8.90 × 10(-4), 7.10 × 10(-4), and 6.65 × 10(-4) mL mol g(-2), respectively). Further substantiation is provided by estimates of the excess binding energy of protein-protein interactions in the condensed phase, obtained from measurements of the PEG-induced liquid-liquid phase separation profiles for the three proteins. The data suggest that enhanced attractive protein-protein interactions, arising from the deamidation of HGS, promote protein aggregation, thereby leading to increased light scattering and opacity over time.

Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions: Part 1. Unfolding, Colloidal Interactions, and Formation of High-Molecular-Weight Aggregates

Monomeric and aggregated states of an IgG1 antibody were characterized under acidic conditions as a function of solution pH (3.5-5.5). A combination of intrinsic/extrinsic fluorescence (FL), circular dichroism, calorimetry, chromatography, capillary electrophoresis, and laser light scattering were used to characterize unfolding, refolding, native colloidal interactions, aggregate structure and morphology, and aggregate dissociation. Lower pH led to larger net repulsive colloidal interactions, decreased thermal stability of Fc and Fab regions, and increased solubility of thermally accelerated aggregates. Unfolding of the Fab domains, and possibly the CH3 domain, was inferred as a key step in the formation of aggregation-prone monomers. High-molecular-weight soluble aggregates displayed nonnative secondary structure, had a semi-rigid chain morphology, and bound thioflavin T (ThT), consistent with at least a portion of the monomer forming amyloid-like structures. Soluble aggregates also formed during monomer refolding under conditions moving from high to low denaturant concentrations. Both thermally and chemically induced aggregates showed similar ThT binding and secondary structural changes, and were noncovalent based on dissociation in concentrated guanidine hydrochloride solutions. Changes in intrinsic FL during chemical versus thermal unfolding suggest a greater degree of structural change during chemical unfolding, although aggregation proceeded through partially unfolded monomers in both cases.

Interactions between Adsorbed Layers of αS1-Casein with Covalently Bound Side Chains: A Self-Consistent Field Study

The effect on the adsorbed layer properties of the modification of alpha S1-casein by covalent bonding with an uncharged polysaccharide side chain has been investigated using lattice-based self-consistent field (SCF) theory. Interactions between two hydrophobic planar surfaces coated by a layer of adsorbed modified alpha S1-casein have been studied as a function of pH and ionic strength. While the interactions of the unmodified alpha S1-casein layers become attractive at high ionic strength, it has been shown that the presence of polysaccharide attachment to the alpha S1-casein molecule can confer net repulsive interactions over a wide range of salt concentration. The disordered protein is represented as a linear flexible polyampholyte with a sequence of hydrophobic, polar, and charged units based on the known alpha S1-casein primary structure. The hydrophilic side chain is attached at various fixed positions along the casein backbone. Different lengths and locations of the attached polysaccharide side chain are examined. Interfacial structures and colloidal stability properties of the system are determined, including the surface-surface interaction potential, the extent of protein bridging, and the distribution of protein segments from the surface under different conditions of pH and ionic strength. It has been found that the covalent bonding of short hydrophilic chains may not only enhance but can also worsen the colloidal stabilizing properties of the modified protein, depending on the position of the attachment.

The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient.

Protein aggregation is commonly observed during protein refolding. To better understand this phenomenon, the intermolecular interactions experienced by a protein during unfolding and refolding are inferred from second virial coefficient (SVC) measurements. It is accepted that a negative SVC is indicative of protein-protein interactions that are attractive, whereas a positive SVC indicates net repulsive interactions. Lysozyme denatured and reduced in guanidinium hydrochloride exhibited a decreasing SVC as the denaturant was diluted, and the SVC approached zero at approximately 3 M GdnHCl. Further dilution of denaturant to renaturation conditions (1.25 M GdnHCl) led to a negative SVC, and significant protein aggregation was observed. The inclusion of 500 mM L-arginine in the renaturation buffer shifted the SVC to positive and suppressed aggregation, thereby increasing refolding yield. The formation of mixed disulfides in the denatured state prior to refolding also increased protein solubility and suppressed aggregation, even without the use of L-arginine. Again, the suppression of aggregation was shown to be caused by a shift from attractive to repulsive intermolecular interactions as reflected in a shift from a negative to a positive SVC value. To the best of our knowledge, this is the first time that SVC data have been reported for renaturation studies. We believe this technique will aid in our understanding of how certain conditions promote renaturation and increase protein solubility, thereby suppressing aggregation. SVC measurements provide a useful link, for protein folding and aggregation, between empirical observation and thermodynamics.

Quantitative Analysis of Fluid Interface–Atomic Force Microscopy

Net repulsive interactions between n-hexadecane and a poly-styrene microsphere in aqueous solutions are measured with atomic force microscopy and interpreted using the augmented Young–Laplace equation. The true separation between probe and fluid interface is implicitly computed from the force–distance data, providing a more accurate description of drop or bubble deformation. Experiments and theoretical arguments demonstrate that a fluid interface stiffens with increasing deformation and is not accurately treated as a Hookean spring. The unexpected stability of the draining aqueous film between hydrophobic bodies in electrolyte solutions is explained primarily by the deformation of the oil drop in response to the applied normal force, as well as the increased hydrodynamic resistance due to the increased drainage area.

Distribution of Distances between Units in Two-Dimensional Excluded-Volume Chains

The distribution of distances between different pairs of units in a two-dimensional linear flexible polymer chain with net repulsive interactions between units (i.e., in the excluded-volume regime) is investigated through an off-lattice Monte Carlo method. Three different cases, according to the position of the pair components (end or interior units), are studied. The results are in excellent agreement with theoretical predictions generated by Coulomb gas techniques and conformal invariance methods. Some of these results, however, differ clearly from the values obtained in previous simulations or from those predicted by e expansion formulas based on the renormalization group theory

The growth modes of vapor-deposited bismuth on a Cu(110) surface

The interactions of vapor-deposited Bi with Cu(110) have been studied with x-ray photoelectron spectroscopy (XPS), thermal desorption mass spectroscopy (TDS), and low-energy electron diffraction (LEED). There is a net repulsive Bi–Bi interaction in the adlayer such that the heat of adsorption decreases from ∼69 to 51 kcal/mol as θBi increases from ∼0.3 to ∼0.7. At low coverages (i.e., long Bi–Bi distances), this repulsive interaction is relatively isotropic, with the Bi forming a quasihexagonal c(2×2) adlayer. At high coverages (i.e., short Bi–Bi distances), the effective Bi–Bi interaction potential is very anisotropic, wherein there are Bi–Bi repulsions parallel to the troughs of the Cu(110) surface, but Bi–Bi attractions across these troughs. This leads to p(n×1) overlayer structures up to completion of the monolayer. Systematic trends in the structural growth modes for metal adlayers showing net repulsive lateral interactions on one-dimensionally corrugated substrates are analyzed. It is found that three types of growth modes can be predicted depending upon a size parameter (the ratio of the metallic diameter of the adatom to the trough–trough separation of the substrate) and the nature of the substrate (fcc vs bcc).

Stabilization of emulsions by fine particles I. Partitioning of particles between continuous phase and oil/water interface

Some of the physicochemical mechanisms governing the stability of oil/water emulsions in the presence of fine particles are investigated. The thermodynamics of particle partitioning between a continuous aqueous phase and the oil /water interfaces of the emulsion droplets is analyzed. An isolated particle with an appropriate three-phase contact angle is trapped in a deep energy well at the interface of an oil/water emulsion droplet. This is consistent with the experimental observations that a close-packed two-dimensional structure of particles is formed on the oil/water interfaces of emulsion droplets stabilized by particles. In equilibrium partitioning the energy depth of a single interfacial particle should be balanced by net repulsive interactions between neighbouring particles on the interface. Electric double layer and short-range salvation and solid elastic forces between the interfacial particles are found to be insufficient to provide the required large repulsive interaction.