Protein Phase Behavior Research Articles

Influence of macromolecular precipitants on phase behavior of monoclonal antibodies.

For the successful application of protein crystallization as a downstream step, a profound knowledge of protein phase behavior in solutions is needed. Therefore, a systematic screening was conducted to analyze the influence of macromolecular precipitants in the form of polyethylene glycol (PEG). First, the influence of molecular weight and concentration of PEG at different pH-values were investigated and analyzed in three-dimensional (3-D) phase diagrams to find appropriate conditions in terms of a fast kinetic and crystal size for downstream processing. In comparison to the use of salts as precipitant, PEG was more suitable to obtain compact 3-D crystals over a broad range of conditions, whereby the molecular weight of PEG is, besides the pH-value, the most important parameter. Second, osmotic second virial coefficients as parameters for protein interactions are experimentally determined with static light scattering to gain a deep insight view in the phase behavior on a molecular basis. The PEG-protein solutions were analyzed as a pseudo-one-compartment system. As the precipitant is also a macromolecule, the new approach of analyzing cross-interactions between the protein and the macromolecule PEG in form of the osmotic second cross-virial coefficient (B23 ) was applied. Both parameters help to understand the protein phase behavior. However, a predictive description of protein phase behavior for systems consisting of monoclonal antibodies and PEG as precipitant is not possible, as kinetic phenomena and concentration dependencies were not taken into account.

Implications of protein polymorphism on protein phase behaviour.

The phase behaviour of small globular proteins is often modeled by approximating them as spherical particles with fixed internal structure. However, changes in the local environment of a protein can lead to changes in its conformation rendering this approximation invalid. We present a simple two-state model in which protein conformation is not conserved and where the high-energy, non-native state is stabilised by pair-wise attractive interactions. The resulting phase behaviour is remarkably complex, non-universal and exhibits re-entrance. The model calculations show a demarcation between a regime where conformational transitioning is largely enslaved by phase separation and one where this is not the case. In the latter regime, which is characterised by a large free energy difference between the native and the non-native state, we deduce that the kinetics of the phase transition strongly depend on the average conformation of the proteins prior to their condensation. For condensation to occur in this regime within a dispersion of native proteins, nucleation of a cluster of proteins in the non-native state is required. We argue that our theory supports the distinction between common phase separation and the nucleated assembly of non-native supramolecular aggregates in protein dispersions.

Determination of protein phase diagrams by microbatch experiments: Exploring the influence of precipitants and pH

Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry. Proteins can either be soluble, crystalline or precipitated. Additionally liquid–liquid phase separation, gelation and skin formation can occur. A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. For lysozyme from chicken egg white, human lysozyme, glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values – pH 3, 5, 7 and 9. Sodium chloride, ammonium sulfate, polyethylene glycol 300 and polyethylene glycol 1000 were used as precipitants. Crystallizing conditions could be found for lysozyme from chicken egg white using sodium chloride, for human lysozyme using sodium chloride or ammonium sulfate and glucose isomerase using ammonium sulfate. PEG caused destabilization of human lysozyme and glucose oxidase solutions or a balance of stabilizing and destabilizing effects for glucose isomerase near the isoelectric point. This work presents a systematic generation and extensive study of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any protein solution.

A comparative study of monoclonal antibodies. 1. Phase behavior and protein-protein interactions.

Protein phase behavior is involved in numerous aspects of downstream processing, either by design as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. This work explores the phase behavior of eight monoclonal antibodies (mAbs) that exhibit liquid-liquid separation, aggregation, gelation, and crystallization. The phase behavior has been studied systematically as a function of a number of factors, including solution composition and pH, in order to explore the degree of variability among different antibodies. Comparisons of the locations of phase boundaries show consistent trends as a function of solution composition; however, changing the solution pH has different effects on each of the antibodies studied. Furthermore, the types of dense phases formed varied among the antibodies. Protein-protein interactions, as reflected by values of the osmotic second virial coefficient, are used to correlate the phase behavior. The primary findings are that values of the osmotic second virial coefficient are useful for correlating phase boundary locations, though there is appreciable variability among the antibodies in the apparent strengths of the intrinsic protein-protein attraction manifested. However, the osmotic second virial coefficient does not provide a clear basis to predict the type of dense phase likely to result under a given set of solution conditions.

Hierarchical organization of chiral rafts in colloidal membranes.

Liquid-liquid phase separation is ubiquitous in suspensions of nanoparticles, proteins and colloids. It has an important role in gel formation, protein crystallization and perhaps even as an organizing principle in cellular biology. With a few notable exceptions, liquid-liquid phase separation in bulk proceeds through the continuous coalescence of droplets until the system undergoes complete phase separation. But when colloids, nanoparticles or proteins are confined to interfaces, surfaces or membranes, their interactions differ fundamentally from those mediated by isotropic solvents, and this results in significantly more complex phase behaviour. Here we show that liquid-liquid phase separation in monolayer membranes composed of two dissimilar chiral colloidal rods gives rise to thermodynamically stable rafts that constantly exchange monomeric rods with the background reservoir to maintain a self-limited size. We visualize and manipulate rafts to quantify their assembly kinetics and to show that membrane distortions arising from the rods' chirality lead to long-range repulsive raft-raft interactions. Rafts assemble into cluster crystals at high densities, but they can also form bonds to yield higher-order structures. Taken together, our observations demonstrate a robust membrane-based pathway for the assembly of monodisperse membrane clusters that is complementary to existing methods for colloid assembly in bulk suspensions. They also reveal that chiral inclusions in membranes can acquire long-range repulsive interactions, which might more generally have a role in stabilizing assemblages of finite size.

Moving through three-dimensional phase diagrams of monoclonal antibodies.

Protein phase behavior characterization is a multivariate problem due to the high amount of influencing parameters and the diversity of the proteins. Single influences on the protein are not understood and fundamental knowledge remains to be obtained. For this purpose, a systematic screening method was developed to characterize the influence of fluid phase conditions on the phase behavior of proteins in three-dimensional phase diagrams. This approach was applied to three monoclonal antibodies to investigate influences of pH, protein and salt concentrations, with five different salts being tested. Although differences exist between the antibodies, this extensive study confirmed the general applicability of the Hofmeister series over the broad parameter range analyzed. The influence of the different salts on the aggregation (crystallization and precipitation) probability was described qualitatively using this Hofmeister series, with a differentiation between crystallization and precipitation being impossible, however.

Competition between Monomeric and Dimeric Crystals in Schematic Models for Globular Proteins

Advances in experimental techniques and in theoretical models have improved our understanding of protein crystallization. However, they have also left open questions regarding the protein phase behavior and self-assembly kinetics, such as why (nearly) identical crystallization conditions can sometimes result in the formation of different crystal forms. Here, we develop a patchy particle model with competing sets of patches that provides a microscopic explanation of this phenomenon. We identify different regimes in which one or two crystal forms can coexist with a low-density fluid. Using analytical approximations, we extend our findings to different crystal phases, providing a general framework for treating protein crystallization when multiple crystal forms compete. Our results also suggest different experimental routes for targeting a specific crystal form, and for reducing the dynamical competition between the two forms, thus facilitating protein crystal assembly.

Phase behavior of the modified-Yukawa fluid and its sticky limit

Simple model systems with short-range attractive potentials have turned out to play a crucial role in determining theoretically the phase behavior of proteins or colloids. However, as pointed out by D. Gazzillo [J. Chem. Phys. 134, 124504 (2011)], one of these widely used model potentials, namely, the attractive hard-core Yukawa potential, shows an unphysical behavior when one approaches its sticky limit, since the second virial coefficient is diverging. However, it is exactly this second virial coefficient that is typically used to depict the experimental phase diagram for a large variety of complex fluids and that, in addition, plays an important role in the Noro-Frenkel scaling law [J. Chem. Phys. 113, 2941 (2000)], which is thus not applicable to the Yukawa fluid. To overcome this deficiency of the attractive Yukawa potential, D. Gazzillo has proposed the so-called modified hard-core attractive Yukawa fluid, which allows one to correctly obtain the second and third virial coefficients of adhesive hard-spheres starting from a system with an attractive logarithmic Yukawa-like interaction. In this work we present liquid-vapor coexistence curves for this system and investigate its behavior close to the sticky limit. Results have been obtained with the self-consistent Ornstein-Zernike approximation (SCOZA) for values of the reduced inverse screening length parameter up to 18. The accuracy of SCOZA has been assessed by comparison with Monte Carlo simulations.

A Small-Angle X-ray Scattering Study of α-helical Bundle-Forming Peptide–Polymer Conjugates in Solution: Chain Conformations

As a new family of soft materials, peptide/protein–polymer conjugates can lead to a wide range of potential biological and nonbiological applications. The performance of these materials depends on the protein structure and phase behavior arising from a balance between the enthalpic interactions of the components and surrounding media as well as the entropic contribution associated with polymer chain deformation. There is a great need to perform structural studies in solution that systematically investigate the polymer chain conformation upon linkage to a peptide or protein so as to evaluate how polymers affect the protein structure of the biomolecule and, consequently, its functionality. Combinations of a range of factors including low contrast, weak scattering signals in dilute solutions as well as difficulties in separating the component scattering contributions, pose significant challenges to structural characterization. Here we present a synchrotron small-angle X-ray scattering (SAXS) study of two model helix bundle forming peptide–polymer conjugates and show that with analytical modeling of the scattering intensity detailed structural information on both peptide structure and polymer conformation can be extracted. The peptide–poly(ethylene glycol) (PEG) conjugates are based on peptides that self-associate to form well-defined 3- or 4-helix bundles and the PEG chain is covalently linked either to the end or the side of the peptide (i.e. end- or side-conjugation). Using a simplified analytical geometrical body form factor model, where the peptide–polymer bundles are modeled as parallel cylinders with attached Gaussian chains, a quantitative description of the scattering behavior can be reached. On the basis of the simplified structural model, the protein tertiary structures, i.e., the α-helix bundle, remains largely intact and maintains its oligomeric state but exhibits slight swelling in solution with respect to the crystal structure. The PEG chain conformation appears to slightly depend on the conjugate architecture. In terms of the chain dimension represented by Rg, the end-conjugated PEG exhibit similar value as compared to free PEG for the molecular weight studied (2 kDa). For the side-conjugates our simple scattering model seems to indicate a systematically slightly lower values for Rg, i.e., a slight compression, in particular for the highest molecular weight (5 kDa). However, considering the limitations of the model and experimental uncertainties, further investigations, such as neutron scattering, is needed to illustrate detailed chain conformation. The present studies can be extended to other peptide–polymer or protein–polymer hybrid systems to extract information on both protein structure and polymer chain conformation. This work will thus provide valuable guidance to understand their structure and phase behavior using X-ray and neutron scattering.

Microfluidics with In-Situ Small-Angle X-Ray Scattering: A Tool to Investigate the Neurofilament Self-Assembly Mechanism

The use of microfluidic chips with in-situ small-angle X-ray scattering (SAXS), offers new interesting possibilities for the study of biomaterials under flow. In first place, the manipulation of fluids allows for an experimental control (e.g. rate of mixing, shear rate, concentration gradients, confinement) that has been previously unavailable, opening the possibility for new experiments. In second place, sample consumption is reduced to the microliter scale, allowing experiments with expensive and rare materials. In third place, the constant flow of material prevents radiation damage (critical for X-ray synchrotron radiation).In this work, we describe an approach to probe the mechanism of self-assembly of mature neurofilaments from its individual protein subunits at physiological ratio (NF-L:NF-M:NF-H of 7:3:2). This process is believed to consist of a series of distinct steps in vitro, involving the formation of several intermediate structures, but its full description has not been reported so far (Janmey PA. et al., 2003, Curr. Opin. Colloid Interface Sci. 8:40-47).The protein solution under strong denaturing conditions (4 and 8 M urea, which prevents the assembly process) is mixed under flow with physiological buffer, leading to a drop in the urea concentration to levels where filament formation is favored. The geometry of the chip allows time-resolved tracking of the assembly process along the main microchannel. The emergence of new structural features with time, typical of larger aggregates, compared to the initial unimer solutions, is observed.Supported by DOE-BES DE-FG02-06ER46314 (dynamic evolution of assemblies) and NSF DMR-1101900 (protein phase behavior). BFBS acknowledges funding from the European Community under a Marie Curie IOF grant (n252701).

Phase behavior of colloids and proteins in aqueous suspensions: Theory and computer simulations

The fluid phase behavior of colloidal suspensions with short-range attractive interactions is studied by means of Monte Carlo computer simulations and two theoretical approximations, namely, the discrete perturbation theory and the so-called self-consistent Ornstein-Zernike approximation. The suspensions are modeled as hard-core attractive Yukawa (HCAY) and Asakura-Oosawa (AO) fluids. A detailed comparison of the liquid-vapor phase diagrams obtained through different routes is presented. We confirm Noro-Frenkel's extended law of scaling according to which the properties of a short-ranged fluid at a given temperature and density are independent of the detailed form of the interaction, but just depend on the value of the second virial coefficient. By mapping the HCAY and AO fluids onto an equivalent square-well fluid of appropriate range at the critical point we show that the critical temperature as a function of the effective range is independent of the interaction potential, i.e., all curves fall in a master curve. Our findings are corroborated with recent experimental data for lysozyme proteins.

Self-assembly of tunable protein suprastructures from recombinant oleosin.

Using recombinant amphiphilic proteins to self-assemble suprastructures would allow precise control over surfactant chemistry and the facile incorporation of biological functionality. We used cryo-TEM to confirm self-assembled structures from recombinantly produced mutants of the naturally occurring sunflower protein, oleosin. We studied the phase behavior of protein self-assembly as a function of solution ionic strength and protein hydrophilic fraction, observing nanometric fibers, sheets, and vesicles. Vesicle membrane thickness correlated with increasing hydrophilic fraction for a fixed hydrophobic domain length. The existence of a bilayer membrane was corroborated in giant vesicles through the localized encapsulation of hydrophobic Nile red and hydrophilic calcein. Circular dichroism revealed that changes in nanostructural morphology in this family of mutants was unrelated to changes in secondary structure. Ultimately, we envision the use of recombinant techniques to introduce novel functionality into these materials for biological applications.

Biofunctionalization and self-interaction chromatography in PDMS microchannels

In this paper we present an experimental protocol for protein immobilization on polydimethylsiloxane (PDMS) polymer surfaces and the subsequent application of a chromatographic PDMS microfluidic chip to measure protein–protein interactions. The PDMS surface modification steps are quantitatively and qualitatively experimentally analyzed using an array of techniques (water contact angle measurement, fluorescence spectroscopy and X-ray photoelectron spectroscopy). The protocol involves PDMS acidic surface activation using a potassium disulfite/potassium peroxidisulfate/acrylic acid mixture, followed by amination with 3-aminopropyl diethoxymethylsilane, followed by glutaraldehyde grafting and subsequent covalent protein binding. The applicability of such a miniaturized PDMS-based microfluidic system has been exemplified by measuring protein–protein interactions in a fast and accurate fashion for three model proteins, namely: hen egg white lysozyme, bovine ribonuclease-A and α-chymotrypsinogen. The protein interaction results align well with existing literature data using different materials and techniques. As the fabrication process for PDMS-based microstructures is relatively cheap, quick and requires limited lab expertise/access to specialized equipments, we consider that the implementation of such a flexible, easy to fabricate, PDMS-based microfluidic system for estimating protein interactions an important step toward quickly mapping protein phase behavior and measuring protein (self/cross) interactions in complex biological systems.

The Effect of Ionic Strength, Temperature, and Pressure on the Interaction Potential of Dense Protein Solutions: From Nonlinear Pressure Response to Protein Crystallization

Understanding the intermolecular interaction potential, V(r), of proteins under the influence of temperature, pressure, and salt concentration is essential for understanding protein aggregation, crystallization, and protein phase behavior in general. Here, we report small-angle x-ray scattering studies on dense lysozyme solutions of high ionic strength as a function of temperature and pressure. We show that the interaction potential changes in a nonlinear fashion over a wide range of temperatures, salt, and protein concentrations. Neither temperature nor protein and salt concentration lead to marked changes in the pressure dependence of V(r), indicating that changes of the water structure dominate the pressure dependence of the intermolecular forces. Furthermore, by analysis of the temperature, pressure, and ionic strength dependence of the normalized second virial coefficient, b2, we show that the interaction can be fine-tuned by pressure, which can be used to optimize b2 values for controlled protein crystallization.

Interactions and phase behavior of a monoclonal antibody

Protein phase behavior is implicated in numerous aspects of downstream processing either by design, as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. An improved understanding of protein phase behavior is, therefore, important for developing rational design strategies for important process steps. This work explores the phase behavior of a monoclonal antibody (mAb), IDEC-152, which exhibits liquid-liquid separation, aggregation, gelation, and crystallization. A systematic study of numerous factors, including the effects of solution composition and pH, has been conducted to explore the phase behavior of this antibody. Phenomena observed include a significant dependence of the cloud point on the cation in sulfate salts and nonmonotonic trends in pH dependence. Additionally, conditions for crystallization of this mAb are reported for the first time. Protein-protein interactions, as determined from the osmotic second virial coefficient, are used to interpret the phase behavior.

Universality of protein reentrant condensation in solution induced by multivalent metal ions

The effective interactions and phase behavior of protein solutions under strong electrostatic coupling conditions are difficult to understand due to the complex charge pattern and irregular geometry of protein surfaces. This distinguishes them from related systems such as DNA or conventional colloids. In this work, we discuss the question of universality of the reentrant condensation (RC) of proteins in solution induced by multivalent counterions, i.e., redissolution on adding further salts after phase separation, as recently discovered (Zhang et al., Phys Rev Lett 2008; 101:148101). The discussion is based on a systematic investigation of five different proteins with different charge patterns and five different multivalent counterions. Zeta potential measurements confirm the effective charge inversion of proteins in the reentrant regime via binding of multivalent counterions, which is supported by Monte Carlo simulations. Charge inversion by trivalent cations requires an overall negative net charge of the protein. Statistical analysis of a representative set of protein sequences reveals that, in theory, this effect could be possible for about half of all proteins. Our results can be exploited for the control of the phase behavior of proteins, in particular facilitating protein crystallization.

Determination of the phase diagram for soluble and membrane proteins.

Methods to efficiently determine the phase behavior of novel proteins have the potential to significantly benefit structural biology efforts. Here, we present protocols to determine both the solubility boundary and the supersolubility boundary for protein/precipitant systems using an evaporation-based crystallization platform. This strategy takes advantage of the well-defined rates of evaporation that occur in this platform to determine the state of the droplet at any point in time without relying on an equilibrium-based end point. The dynamic nature of this method efficiently traverses phase space along a known path, such that a solubility diagram can be mapped out for both soluble and membrane proteins while using a smaller amount of protein than what is typically used in optimization screens. Furthermore, a variation on this method can be used to decouple crystal nucleation and growth events, so fewer and larger crystals can be obtained within a given droplet. The latter protocol can be used to rescue a crystallization trial where showers of tiny crystals were observed. We validated both of the protocols to determine the phase behavior and the protocol to optimize crystal quality using the soluble proteins lysozyme and ribonuclease A as well as the membrane protein bacteriorhodopsin.

Phase transitions of folded proteins

Proteins exhibit a rich phase behavior. Even omitting the amyloid structures formed after partial protein unfolding, proteins form crystals, polymers, and other solid aggregates, as well as dense liquids and gels. Some of these condensed phases underlie pathological conditions, others play a crucial role in the biological function of the respective protein or are an essential part of its laboratory or industrial processing. In this review, we first discuss the thermodynamic characteristics of the solution and the interactions between protein molecules in solution, which underlie the protein phase behavior. We highlight the role of water structured at the protein molecular surface as a main contributor to the free energy of the phase transition. We define the driving force for phase transformations. We then summarize the fundamentals and review recent findings on the kinetics of nucleation of dense liquid droplets and crystals. We define the transition from nucleation to spinodal decomposition for these two phase transitions. We review the two-step mechanism of protein crystal nucleation, in which mesoscopic metastable protein clusters serve as precursors to the ordered crystal nuclei. Lastly, we discuss the mechanisms of growth of crystals: the generation of new crystal layers, the pathways of the molecules from the solution into the growth sites, the density of the growth sites and the factors, which determine the activation barriers for association of the molecules to the growth sites.

Effects of oxidase and protease treatments on the breadmaking functionality of a range of gluten-free flours

In this study, the application of glucose oxidase and protease commercial preparations was investigated in order to evaluate their impact on the breadmaking performance of four different gluten-free flours (buckwheat, corn, sorghum and teff). Bread formulas were developed without addition of hydrocolloids in order to avoid synergistic effects. Glucose oxidase improved corn (CR) and sorghum (SG) bread quality by increasing specific volume (P < 0.05) and reducing collapsing at the top. The improvements could be related to protein polymerization which resulted in enhanced continuity of the protein phase and elastic-like behavior of CR and SG batters. No significant effects were detected on buckwheat (BW) and teff breads. On the other hand, protease treatment had detrimental effects on the textural quality of BW and SG breads. The effects were related to protein degradation resulting in increased liquid-like behaviour of BW and SG batters. Overall, the results of this study suggest that protein polymerisation can improve the breadmaking performance of gluten-free flours by enhancing elastic-like behaviour of batters. However, the protein source is a key element determining the impact of the enzymes. In the absence of hydrocolloids, protein structures are important to ensure the textural quality of these types of breads.

Comparative Effects of Salt, Organic, and Polymer Precipitants on Protein Phase Behavior and Implications for Vapor Diffusion

Salts, polymers, and organic precipitants commonly used to crystallize proteins share the ability to induce attractive protein−protein interactions, and eventually lead to the formation of crystals. Their effects on protein phase behavior are investigated here. First, the ovalbumin phase diagram at pH 7 was determined separately in ammonium sulfate, poly(ethylene glycol) (PEG) 8000, and 2-methyl-2,4-pentanediol (MPD) solutions. Increasing concentrations of each of these three additives lead to well-defined phase separations that have the same characteristic trends, but differ in their physical appearance. Second, the phase diagrams of ovalbumin in ammonium sulfate and ribonuclease A in MPD were determined at pH 6, conditions under which the two proteins crystallize. The general shape of the phase diagram and the formation kinetics of the different phases are similar for both proteins, suggesting that the main characteristics of the phase behavior are independent of the physical origin of the attraction be...