Protein-polyelectrolyte Complexes Research Articles

Membrane-Free Lateral Flow Assay with the Active Control of Fluid Transport for Ultrasensitive Cardiac Biomarker Detection.

Membrane-based lateral flow immunoassays (LFAs) have been employed as early point-of-care (POC) testing tools in clinical settings. However, the varying membrane properties, uncontrollable sample transport in LFAs, visual readout, and required large sample volumes have been major limiting factors in realizing needed sensitivity and desirable precise quantification. Addressing these challenges, we designed a membrane-free system in which the desirable three-dimensional (3D) structure of the detection zone is imitated and used a small pump for fluid flow and fluorescence as readout, all the while maintaining a one-step assay protocol. A hydrogel-like protein-polyelectrolyte complex (PPC) within a polyelectrolyte multilayer (PEM) was developed as the test line by complexing polystreptavidin (pSA) with poly(diallyldimethylammonium chloride) (PDDA), which in turn was layered with poly(acrylic acid) (PAA) resulting in a superior 3D streptavidin-rich test line. Since the remainder of the microchannel remains material-free, good flow control is achieved, and with the total volume of 20 μL, 7.5-fold smaller sample volumes can be used in comparison to conventional LFAs. High sensitivity with desirable reproducibility and a 20 min total assay time were achieved for the detection of NT-proBNP in plasma with a dynamic range of 60-9000 pg·mL-1 and a limit of detection of 56 pg·mL-1 using probe antibody-modified fluorescence nanoparticles. While instrument-free visual detection is no longer possible, the developed lateral flow channel platform has the potential to dramatically expand the LFA applicability, as it overcomes the limitations of membrane-based immunoassays, ultimately improving the accuracy and reducing the sample volume so that finger-prick analyses can easily be done in a one-step assay for analytes present at very low concentrations.

Complexation of poly(methacrylic acid) star polyelectrolytes with lysozyme

Protein-polyelectrolyte complexes are of great interest due to their applications in both biological and industrial systems. This study focuses on investigating the electrostatic complexation between (hen egg white) lysozyme (LYZ), a well-studied enzyme, and two star-shaped poly(methacrylic acid) (PMAA) polymers with different arm molar masses, namely S-PMAA5K and S-PMAA10K. Depending on the mixing ratio of the two components (protein/star polyelectrolyte) either stable solutions were formed or macroscopic phase separation, i.e. coacervation, was observed. Dynamic and electrophoretic light scattering results reveal that the overall physicochemical properties of the complexes and the strength of the interaction depends on the ratio of the two components as well as on the molar mass of the polymer, with the S-PMAA10K series exhibiting stronger interactions compared to S-PMAA5K. Additional information regarding the molar mass of the star polyelectrolytes and the corresponding complexes with lysozyme were acquired by analytical ultracentrifugation. The morphology of the complexes was further elucidated through atomic force and cryo-transmission electron microscopy showing globular raspberry-like objects. Moreover, fluorescence and infrared spectroscopic techniques were employed to explore potential conformational changes of the protein upon complexation, with both techniques indicating that the protein does not undergo significant structural changes. Furthermore, UV–Vis spectroscopy was utilized to quantify the amount of protein present in the supernatant of the coacervated complex solutions, thus allowing the quantification of the unbound protein remaining in the solution after complex formation.

Reversible protein complexes as a promising avenue for the development of high concentration formulations of biologics

High concentration formulations have become an important pre-requisite in the development of biological drugs, particularly in the case of subcutaneous administration where limited injection volume negatively affects the administered dose. In this study, we propose to develop high concentration formulations of biologics using a reversible protein-polyelectrolyte complex (RPC) approach. First, the versatility of RPC was assessed using different complexing agents and formats of therapeutic proteins, to define the optimal conditions for complexation and dissociation of the complex. The stability of the protein was investigated before and after complexation, as well as upon a 4-week storage period at various temperatures. Subsequently, two approaches were selected to develop high concentration RPC formulations: first, using up-concentrated RPC suspensions in aqueous buffers, and second, by generating spray-dried RPC and further resuspension in non-aqueous solvents. Results showed that the RPC concept is applicable to a wide range of therapeutic protein formats and the complexation-dissociation process did not affect the stability of the proteins. High concentration formulations up to 200 mg/mL could be achieved by up-concentrating RPC suspensions in aqueous buffers and RPC suspensions in non-aqueous solvents were concentrated up to 250 mg/mL. Although optimization is needed, our data suggests that RPC may be a promising avenue to achieve high concentration formulations of biologics for subcutaneous administration.

Effects of Control Factors on Protein-Polyelectrolyte Complex Coacervation.

Protein-polyelectrolyte complex coacervation is of particular interest for mimicking intracellular phase separation and organization. Yet, the challenge arises from regulating the coacervation due to the globular structure and anisotropic distributed charges of protein. Herein, we fully investigate the different control factors and reveal their effects on protein-polyelectrolyte coacervation. We prepared mixtures of BSA (bovine serum albumin) with different cationic polymers, which include linear and branched polyelectrolytes covering different spacer and charge groups, chain lengths, and polymer structures. With BSA-PDMAEMA [poly(N,N-dimethylaminomethyl methacrylate)] as the main investigated pair, we find that the moderate pH and ionic strength are essential for the adequate electrostatic interaction and formation of coacervate droplets. For most BSA-polymer mixtures, excess polyelectrolytes are required to achieve the full complexation, as evidenced by the deviated optimal charge mixing ratios from the charge stoichiometry. Polymers with longer chains or primary amine groups and a branched structure endow a strong electrostatic interaction with BSA and cause a bigger charge ratio deviation associated with the formation of solid-like coacervate complexes. Nevertheless, both the liquid- and solid-like coacervates hardly interrupt the BSA structure and activity, indicating the safe encapsulation of proteins by the coacervation with polyelectrolytes. Our study validates the crucial control of the diverse factors in regulating protein-polyelectrolyte coacervation, and the revealed principles shall be instructive for establishing other protein-based coacervations and boosting their potential applications.

Surface Properties of Protein-Polyelectrolyte Solutions. Impact of Polyelectrolyte Hydrophobicity.

The strong influence of an amphiphilic polyelectrolyte, poly(N,N-diallyl-N-hexyl-N-methylammonium chloride), on the surface properties of solutions of globular proteins (lysozyme, β-lactoglobulin, bovine serum albumin, and green fluorescent protein) depends on the protein structure and allows elucidation of the contribution of hydrophobic interactions in the protein-polyelectrolyte complex formation at the liquid-gas interface. At the beginning of adsorption, the surface properties are determined by the unbound amphiphilic component, but the influence of the protein-polyelectrolyte complexes of high surface activity increases at the approach to equilibrium. The kinetic dependencies of the dilational dynamic surface elasticity with one or two local maxima give a possibility to distinguish clearly between different steps of the adsorption process and to trace the formation of the distal region of the adsorption layer. The conclusions from the surface rheological data are corroborated by ellipsometric and tensiometric results.

Biomolecular Complexation on the "Wrong Side": A Case Study of the Influence of Salts and Sugars on the Interactions between Bovine Serum Albumin and Sodium Polystyrene Sulfonate.

In the protein purification, drug delivery, food industry, and biotechnological applications involving protein–polyelectrolyte complexation, proper selection of co-solutes and solution conditions plays a crucial role. The onset of (bio)macromolecular complexation occurs even on the so-called “wrong side” of the protein isoionic point where both the protein and the polyelectrolyte are net like-charged. To gain mechanistic insights into the modulatory role of salts (NaCl, NaBr, and NaI) and sugars (sucrose and sucralose) in protein–polyelectrolyte complexation under such conditions, interaction between bovine serum albumin (BSA) and sodium polystyrene sulfonate (NaPSS) at pH = 8.0 was studied by a combination of isothermal titration calorimetry, fluorescence spectroscopy, circular dichroism, and thermodynamic modeling. The BSA–NaPSS complexation proceeds by two binding processes (first, formation of intrapolymer complexes and then formation of interpolymer complexes), both driven by favorable electrostatic interactions between the negatively charged sulfonic groups (−SO3–) of NaPSS and positively charged patches on the BSA surface. Two such positive patches were identified, each responsible for one of the two binding processes. The presence of salts screened both short-range attractive and long-range repulsive electrostatic interactions between both macromolecules, resulting in a nonmonotonic dependence of the binding affinity on the total ionic strength for both binding processes. In addition, distinct anion-specific effects were observed (NaCl < NaBr < NaI). The effect of sugars was less pronounced: sucrose had no effect on the complexation, but its chlorinated analogue, sucralose, promoted it slightly due to the screening of long-range repulsive electrostatic interactions between BSA and NaPSS. Although short-range non-electrostatic interactions are frequently mentioned in the literature in relation to BSA or NaPSS, we found that the main driving force of complexation on the “wrong side” are electrostatic interactions.

Both Charge-Regulation and Charge-Patch Distribution Can Drive Adsorption on the Wrong Side of the Isoelectric Point.

The mechanism of protein-polyelectrolyte complexation on the wrong side of the isoelectric point has long puzzled researchers. Two alternative explanations have been proposed in the literature: (a) the charge-patch (CP) mechanism, based on the inhomogeneous distribution of charges on the protein, and (b) the charge-regulation (CR) mechanism, based on the variable charge of weak acid and base groups, which may invert the protein charge in the presence of another highly charged object. To discern these two mechanisms, we simulated artificially constructed short peptides, containing acidic and basic residues, arranged in a blocklike or alternating sequence. Our simulations of these peptides, interacting with polyelectrolytes, showed that charge patch and charge regulation alone can both lead to adsorption on the wrong side of the pI value. Their simultaneous presence enhances adsorption, whereas their absence prevents adsorption. Our simulation results were rationalized by following the variation of the charge regulation capacity and dipole moments of these peptides with the pH. Specifically for lysozyme, we found that charge patch prevails at physiological pH, whereas charge regulation prevails near the pI, thereby explaining seemingly contradicting conclusions in the literature. By applying the same approach to other proteins, we developed a general framework for assessing the role of the CP and CR mechanisms in existing case studies and for predicting how various proteins interact with polyelectrolytes at different pH values.

Lowering the viscosity of a high-concentration antibody solution by protein–polyelectrolyte complex

High-concentration and low-viscosity antibody formulations are necessary when administering these solutions subcutaneously (SC) due to limitations on injection volume. Here we show a method to decrease the viscosity of monoclonal antibody solution by protein-polyelectrolyte complex (PPC) with poly-l-glutamic acid (polyE). The viscosity of omalizumab solutions was 90cP at the concentration of 150mg/mL. In the presence of 20-50mM polyE, the viscosity of PPC solution of 150mg/mL omalizumab dramatically decreased below 10cP due to the formation of crowded solution. The crowded state of PPC, named aggregated PPC (A-PPC), contained water droplets with a diameter of 10μm or larger with low antibody concentrations. In the presence of 60mM or more polyE, the omalizumab solution was transparent with the viscosity of 40cP or less, named soluble PPC (S-PPC). More importantly, the solutions of both A-PPC and S-PPC were fully redissolved by the addition of phosphate saline buffer confirmed by secondary structure, the amount of aggregates, and binding activity to antigen.

Особенности термоагрегации бычьего сывороточного альбумина в присутствии сильных полиэлектролитов

The influence of temperature on bovine serum albumin (BSA) aggregation in aqueous solutions in the presence of poly-N,N-dimethyl-N,N-diallylammonium chloride (PDMDAAC) and the sodium salt of carboxymethylcellulose (CMC) was studied. It was shown that protein-polyelectrolyte complexes (PPC) form because of macromolecular reactions that are stabilized mainly by electrostatic forces. To characterize the PPC composition the φ parameter was used. This parameter is defined as the ratio of the concentration of ionic groups of polyelectrolyte per protein molecules. It was studied that when in an interpolyelectrolyte reaction, a sufficiently high degree of transformation occurs the polymer electrolyte initiates aggregation of protein molecules. As the temperature increases, the initiating role of the polymer electrolyte increases due to an increase in the intensity of hydrophobic interactions. Using the method of spectrophotometry, it was found that, depending on the nature of the polymer electrolyte, insoluble complexes of bovine serum albumin are formed when the pH parameter is above or below the isoelectric point of the protein, when its macromolecules are negatively or positively charged. In the presence of poly-N,N-dimethyl-N,N-diallylammonium chloride, the intensive formation of aggregates and their rapid precipitation in the form of flakes at pH &gt; 7.0 was observed when the temperature increased to 60 °C. The maximum yield of the product of the interpolyelectrolyte reaction bovine serum albumin – sodium salt of carboxymethylcellulose was detected at pH ≤ 4.0. A temperature increase up to 60 °C, in this case, was not accompanied by intensive flocculation. Under optimal composition and interaction conditions, the degree of transformation in the BSA – PDMDAAX and BSA – CMC reactions is ~0.93 and 0.9, respectively, and decreases by ~5-7% with an increase in temperature to 60 °C. It was shown that for the same BOD composition (the ratio of components in the [CMC]/[BSA] complex = 0.1 g/g), an increase in temperature from 25 to 60 °C leads to the formation of particles that increase in size from 1 mcm to 5 mcm. The temperature increase leads to a change in composition of BOD, corresponding to its maximum output as a interpolyelectrolyte reactions product: for complex with PDMDAAC at T = 25, 40 and 60 °C, the φ value is 70, 60, 15; for the complex with the CMC – 60, 50, 20.

Effect of Protein Surface Charge Distribution on Protein–Polyelectrolyte Complexation

Charge anisotropy or the presence of charge patches at protein surfaces has long been thought to shift the coacervation curves of proteins and has been used to explain the ability of some proteins to coacervate on the "wrong side" of their isoelectric point. This work makes use of a panel of engineered superfolder green fluorescent protein mutants with varying surface charge distributions but equivalent net charge and a suite of strong and weak polyelectrolytes to explore this concept. A patchiness parameter, which assessed the charge correlation between points on the surface of the protein, was used to quantify the patchiness of the designed mutants. Complexation between the polyelectrolytes and proteins showed that the mutant with the largest patchiness parameter was the most likely to form complexes, while the smallest was the least likely to do so. The patchiness parameter was found to correlate well with the phase behavior of the protein-polymer mixtures, where both macrophase separation and the formation of soluble aggregates were promoted by increasing the patchiness depending on the polyelectrolyte with which the protein was mixed. Increasing total charge and increasing strength of the polyelectrolyte promote interactions for oppositely charged polyelectrolytes, while charge regulation is also key to interactions for similarly charged polyelectrolytes, which must interact selectively with oppositely charged patches.

INTERACTION OF NATURAL AND SYNTHETIC POLYELECTROLYTES WITH BOVINE SERUM ALBUMIN

Interaction of a number of natural and synthetic polyelectrolytes (PE): chitosan, poly‒N,N‒dimethyl‒N,N‒diallylammonium chloride, sodium carboxymethylcellulose and aromatic copolyamide, synthesized on the bases of dichloranhydride of isophthalic acid and two diamines (4,4’‒(2,2’‒sodium disulfonate)‒diaminodephenyl, 4,4’‒(2,2’‒dicarboxylic acid)‒diaminodiphenylmethyl) and bovine serum albumin (BSA) in aqueous solutions were studied. It was shown that as a result of macromolecular reactions protein‒polyelectrolyte complexes (PPC) forms, stabilized mainly by electrostatic forces. To characterize their composition we used the value of parameter φ, defined as an amount of ionic groups of polyelectrolytes calculated per macromolecule of protein. Using spectrophotometry and conductometry it was established that the composition of PPC in the studied systems when components are mixed at optimal conditions corresponds to the value of φ ~30 – 90. The conditions for the existence of insoluble PPC were determined. In the case of precipitation of poly-N,N‒dimethyl‒N,N‒diallylammonium chloride and chitosan in protein – polyelectrolyte reactions the maximum yield of the product is observed at pH ≥ 7. The introduction of sodium carboxymethylcellulose and aromatic copolyamide into the interaction with protein accompanied by shifting the range of PPC existence into the acidic region: the maximum yield of the product is observed at рН ≤ 4. The size of the formed complex particles ranges from 10 nm ([PE]/[BSA] = 0.01 – 0.05 g/g) to ~ 1.0 ‒ 5.5 μm ([PE]/[BSA] = 0.1 – 0.35 g/g). In case of relatively short‒chained aromatic copolyamide, containing significant amount of non‒ionized carboxylic groups, the ratio of micron size particles is about 5%. The presence of sulfonate and carboxylic groups in the composition of copolyamide gives an additional opportunity to regulate the conversation degree in the interpolyelectrolyte reactions, thus also implying the structure and the composition of the formed complexes.

Protein-Polyelectrolyte Complexes and Micellar Assemblies.

In this review, we highlight the recent progress in our understanding of the structure, properties and applications of protein–polyelectrolyte complexes in both bulk and micellar assemblies. Protein–polyelectrolyte complexes form the basis of the genetic code, enable facile protein purification, and have emerged as enterprising candidates for simulating protocellular environments and as efficient enzymatic bioreactors. Such complexes undergo self-assembly in bulk due to a combined influence of electrostatic interactions and entropy gains from counterion release. Diversifying the self-assembly by incorporation of block polyelectrolytes has further enabled fabrication of protein–polyelectrolyte complex micelles that are multifunctional carriers for therapeutic targeted delivery of proteins such as enzymes and antibodies. We discuss research efforts focused on the structure, properties and applications of protein–polyelectrolyte complexes in both bulk and micellar assemblies, along with the influences of amphoteric nature of proteins accompanying patchy distribution of charges leading to unique phenomena including multiple complexation windows and complexation on the wrong side of the isoelectric point.

Белок-полиэлектролитные комплексы. Часть 3. Комплексы бычьего сывороточного альбумина с сульфонатсодержащими ароматическими поли- и сополиамидами

The interaction of bovine serum albumin in aqueous solution with sulfonate-containing poly- and copolyamides were studied. It was shown that, as a result of macromolecular reactions protein-polyelectrolyte complexes forms, stabilized mainly by electrostatic forces. To characterize the protein-polyelectrolyte complexes composition the r parameter used, which is defines as the ratio of mass concentration of polyelectrolyte and protein. It was shown that the main factor determining the composition and structure of forming protein-polyelectrolyte complexes is the degree of ionization of functional groups, interacting in the polyelectrolyte reaction that is determined by the nature of those groups and the pH of the solution. The presen ce of sulfonate and carboxylic groups in the copolyamide composition gives an extra opportunity to regulate the protein-polyelectrolyte interactions. Using spectrophotometry were established that, in the studied system when the aromatic polyamide and bovine serum albumin are mixed at optimal pH conditions (рН &lt; 4.9), complexes are formed, the composition of which corresponds to the value of r ~ 0.15 g/g. The degree of conversation in protein-polyelectrolyte reactions is close to 0.8. The size of the formed particles was about 2.2 μm. In the case of aromatic copolyamides that contain both sulfonate and carboxylic groups, an increase in concentration of carboxylic groups to 42 mol. % leads to a shift of the optical density maximum on the curve of turbidimetric titration in to the higher r values (~ 0.18 g/g) at pH = 3.5, when the carboxylic groups are non-ionized. The size of the formed complex particles was about ~ 150.0 nm, the fraction of micron-sized particles is about 5%. The degree of released protein is based on the conditions under which reaction take place and varies from 93 to 99%. The result obtained during this work can serve as a base for the effective methods of isolation and purifying of the target proteins development.

Macro- and Microphase Separated Protein-Polyelectrolyte Complexes: Design Parameters and Current Progress.

Protein-containing polyelectrolyte complexes (PECs) are a diverse class of materials, composed of two or more oppositely charged polyelectrolytes that condense and phase separate near overall charge neutrality. Such phase-separation can take on a variety of morphologies from macrophase separated liquid condensates, to solid precipitates, to monodispersed spherical micelles. In this review, we present an overview of recent advances in protein-containing PECs, with an overall goal of defining relevant design parameters for macro- and microphase separated PECs. For both classes of PECs, the influence of protein characteristics, such as surface charge and patchiness, co-polyelectrolyte characteristics, such as charge density and structure, and overall solution characteristics, such as salt concentration and pH, are considered. After overall design features are established, potential applications in food processing, biosensing, drug delivery, and protein purification are discussed and recent characterization techniques for protein-containing PECs are highlighted.

Effect of additives on liquid droplet of protein-polyelectrolyte complex for high-concentration formulations.

Liquid droplets of protein-polyelectrolyte complexes (PPCs) have been developed as a new candidate for stabilization and concentration of protein drugs. However, it remains unclear whether additives affect the precipitation and redissolution yields of PPCs. In the present study, we investigated the PPC formation of human immunoglobulin G (IgG) and poly-L-glutamic acid (polyE) in the presence of various additives that have diverse effects, such as protein stabilization. Alcohols, including ethanol, successfully increased the PPC precipitation yield to over 90%, and the PPCs formed were completely redissolved at physiological ionic strength. However, poly(ethylene glycol), sugars, and amino acids did not improve the precipitation and redissolution yields of PPCs over those observed when no additives were included. Circular dichroism spectrometry showed that the secondary structure of polyE as well as electrostatic interactions play important roles in increasing the PPC precipitation yield when ethanol is used as an additive. The maximum concentration of IgG reached 100 mg/ml with the use of ethanol, which was 15% higher efficiency of the protein yield after precipitation and redissolution than that in the absence of additives. Thus, the addition of a small amount of ethanol is effective for the concentration and stabilization of precipitated PPCs containing IgG formulations.

Control of Aggregation, Coaggregation, and Liquid Droplet of Proteins Using Small Additives.

This review article presents the concepts of aggregates, coaggregates, and a liquid droplet of proteins and compares the concentrated states in the presence of small additives to control their formation and dissociation. The aggregates composed of single protein molecules result mainly from hydrophobic interactions between unfolded protein molecules. Thus, the aggregation of protein can be effectively suppressed by small additives that increase the solubility of hydrophobic solutes, typically arginine (Arg) and chaotropes. In contrast, coaggregation that is composed of two or more types of proteins results from both hydrophobic and attractive electrostatic interactions between even partially unfolded protein molecules. Accordingly, coaggregation is more controllable than simple aggregation using various types of small additives, such as ions and osmolytes, as well as Arg and chaotropes. The liquid droplets of proteins observed in living cells and a protein-polyelectrolyte complex (PPC) undergo liquid-liquid phase separation driven only by electrostatic interactions. Thus, the liquid droplets and PPCs are redissolved when the concentration of ions is increased. The properties of electrostatic or hydrophobic interactions, solid-like or liquid-like states, and apparently spherical and amorphous structures are simple but valuable criteria that can be used to control protein aggregation and condensation using small additives.

Integrating Proteins in Layer-by-Layer Assemblies Independently of their Electrical Charge.

Layer-by-layer (LbL) assembly is an attractive method for protein immobilization at interfaces, a much wanted step for biotechnologies and biomedicine. Integrating proteins in LbL thin films is however very challenging due to their low conformational entropy, heterogeneous spatial distribution of charges, and polyampholyte nature. Protein-polyelectrolyte complexes (PPCs) are promising building blocks for LbL construction owing to their standardized charge and polyelectrolyte (PE) corona. In this work, lysozyme was complexed with poly(styrenesulfonate) (PSS) at different ionic strengths and pH values. The PPCs size and electrical properties were investigated, and the forces driving complexation were elucidated, in the light of computations of polyelectrolyte conformation, with a view to further unravel LbL construction mechanisms. Quartz crystal microbalance and atomic force microscopy were used to monitor the integration of PPCs compared to the one of bare protein molecules in LbL assemblies, and colorimetric assays were performed to determine the protein amount in the thin films. Layers built with PPCs show higher protein contents and hydration levels. Very importantly, the results also show that LbL construction with PPCs mainly relies on standard PE-PE interactions, independent of the charge state of the protein, in contrast to classical bare protein assembly with PEs. This considerably simplifies the incorporation of proteins in multilayers, which will be beneficial for biosensing, heterogeneous biocatalysis, biotechnologies, and medical applications that require active proteins at interfaces.

Liquid Droplet of Protein-Polyelectrolyte Complex for High-Concentration Formulations

The formulation of high-concentration protein solutions is a challenging issue for achieving subcutaneous administration. Previously, we developed a method of precipitation-redissolution using polyelectrolyte as a precipitant to produce protein solutions at high concentrations. However, the redissolution yield of proteins was insufficient. This study aims to optimize the solution conditions for practical applications by combining IgG and poly-l-(glutamic acid) (polyE). A systematic analysis of solution pH and polyE size conditions revealed that an acidic condition favors precipitation, whereas neutral pH values are more effective for the redissolution. We find that the optimal size for polyE ranged from 15,000 to 50,000. This slight modification in the procedure in comparison with previous studies increased the precipitation and redissolution yields to nearly 100%, without irreversible protein denaturation. The fully reversible IgG-polyE complex formed as a droplet structure, which is similar to a condensate of liquid-liquid phase separation. The droplet structure plays an indispensable role in the salt-induced, redissolved state, which is pertinent to the new application that takes advantage of the methods to produce highly concentrated protein solutions.

Influence of protein charge patches on the structure of protein-polyelectrolyte complexes.

We employ a combination of the single chain in mean field simulation approach with the solution of Poisson's equation to study the influence of charge heterogeneities on the structure of protein-polyelectrolyte complexes. By adopting a coarse-grained model of representing proteins as charged nanoparticles, we studied the influence of the pattern of charge heterogeneities, net charge, ratio of positive to negative charges on the patches, and the volume fraction of the particles on the structural and aggregation characteristics of proteins in polyelectrolyte solutions. Our results demonstrate that the pattern of charge heterogeneities can exert a significant influence on the resulting characteristics of the aggregates, in some cases leading to a transformation from polymer-bridged complexes into direct particle aggregates driven by the attraction between oppositely charged patches.

Computationally Mapping pKa Shifts Due to the Presence of a Polyelectrolyte Chain around Whey Proteins.

Experimental studies have shown the formation of soluble complexes in the pure repulsive Coulombic regime even when the net charges of the protein and the polyelectrolyte have the same sign ( De Kruif et al. Curr. Opin. Colloid Interface Sci. 2004 , 9 , 340 ; De Vries et al. J. Chem. Phys. 2003 , 118 , 4649 ; Grymonpre et al. Biomacromolecules 2001 , 2 , 422 ; Hattori et al. Langmuir 2000 , 16 , 9738 ). This attractive phenomenon has often been described as "complexation on the wrong side of pI". While one theory assumes the existence of "charged patches" on the protein surface from ion-dipole interactions, thus allowing a polyelectrolyte to bind to an oppositely heterogeneous charged protein region, another theoretical view considers the induced-charge interactions to be the dominant factor in these complexations. This charge regulation mechanism can be described by proton fluctuations resulting from mutual rearrangements of the distributions of the charged groups, due to perturbations of the acid-base equilibrium. Using constant-pH Monte Carlo simulations and several quantitative and visual analysis tools, we investigate the significance of each of these interactions for two whey proteins, α-lactalbumin (α-LA) and lysozyme (LYZ). Through physical chemistry parameters, free energies of interactions, and the mapping of amino acid pKa shifts and polyelectrolyte trajectories, we show the charge regulation mechanism to be the most important contributor in protein-polyelectrolyte complexation regardless of pH, dipole moment, and protein capacitance in a low salt regime.