Antibody association in solution: cluster distributions and mechanisms.

Understanding factors that affect the clustering and association of antibodies molecules in solution is critical to their development as therapeutics. For 19 different monoclonal antibody (mAb) solutions, we measured the viscosities, the second virial coefficients, the Kirkwood-Buff integrals, and the cluster distributions of the antibody molecules as functions of protein concentration. Solutions were modeled using the statistical-physics Wertheim liquid-solution theory, representing antibodies as Y-shaped molecular structures of seven beads each. We found that high-viscosity solutions result from more antibody molecules per cluster. Multi-body properties such as viscosity are well predicted experimentally by the 2-body Kirkwood-Buff quantity, G22, but not by the second virial coefficient, B22, and well-predicted theoretically from the Wertheim protein-protein sticking energy. Weakly interacting antibodies are rate-limited by nucleation; strongly interacting ones by propagation. This approach gives a way to relate micro to macro properties of solutions of associating proteins.

Protein Association in Solution: Statistical Mechanical Modeling.

Protein molecules associate in solution, often in clusters beyond pairwise, leading to liquid phase separations and high viscosities. It is often impractical to study these multi-protein systems by atomistic computer simulations, particularly in multi-component solvents. Instead, their forces and states can be studied by liquid state statistical mechanics. However, past such approaches, such as the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, were limited to modeling proteins as spheres, and contained no microscopic structure-property relations. Recently, this limitation has been partly overcome by bringing the powerful Wertheim theory of associating molecules to bear on protein association equilibria. Here, we review these developments.

Biophysical Principles Emerging from Experiments on Protein-Protein Association and Aggregation.

Protein-protein association and aggregation are fundamental processes that play critical roles in various biological phenomena, from cellular signaling to disease progression. Understanding the underlying biophysical principles governing these processes is crucial for elucidating their mechanisms and developing strategies for therapeutic intervention. In this review, we provide an overview of recent experimental studies focused on protein-protein association and aggregation. We explore the key biophysical factors that influence these processes, including protein structure, conformational dynamics, and intermolecular interactions. We discuss the effects of environmental conditions such as temperature, pH and related buffer-specific effects, and ionic strength and related ion-specific effects on protein aggregation. The effects of polymer crowders and sugars are also addressed. We list the techniques used to study aggregation. We analyze emerging trends and challenges in the field, including the development of computational models and the integration of multidisciplinary approaches for a comprehensive understanding of protein-protein association and aggregation. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The Effect of Arginine on the Phase Stability of Aqueous Hen Egg-White Lysozyme Solutions.

The effect of arginine on the phase stability of the hen egg-white lysozyme (HEWL) has been studied via molecular dynamics computer simulations, as well as experimentally via cloud-point temperature determination. The experiments show that the addition of arginine increases the stability of the HEWL solutions. The computer simulation results indicate that arginine molecules tend to self-associate. If arginine residues are located on the protein surface, the free arginine molecules stay in their vicinity and prevent the way protein molecules "connect" through them to form clusters. The results are not sensitive to a particular force field and suggest a possible microscopic mechanism of the stabilizing role of arginine as an excipient.

The Role of Buffers in Wild-Type HEWL Amyloid Fibril Formation Mechanism: A Methodological Approach.

The amyloidophilic dyes thioflavin T and Congo red are small, yet powerful, molecules that allow the in vitro and in vivo detection of amyloid fibrils in protein solutions. Even though Congo red and thioflavin T binding assays are widespread techniques for unveiling amyloid fibers and are gradually replacing the more demanding X-ray diffraction method, handling samples containing amyloid fibrils is still challenging and can lead to false-positive/negative results. Here we describe a relatively straightforward procedure of preparing hen egg-white lysozyme amyloid fibrils in different buffer solutions and their detection with thioflavin T and Congo red, supported by an indispensable method for determining the secondary structure of proteins - circular dichroism.

The mechanism of self-association of human γ-D crystallin from molecular dynamics simulations.

The aggregation of human γ-D crystallin is associated with the age-onset cataract formation. Here, we extensively investigated the self-association mechanism of human γ-D crystallin through molecular dynamics computer simulations. By mutating the protein surface we found that electrostatic interactions between charged amino acids play a crucial role in its self-association. We have confirmed the two-fold role of arginine molecules. If they are located as residues on the protein surface they can initiate protein contacts and contribute to their stickiness with noteworthy hydrophobic interactions through stacking of their methylene groups. But if they are added as free arginine in the protein solution they can also stabilize it, by associating with the protein surface and also with themselves to form effective inter-protein spacers that obstruct protein aggregation.

BioMThermDB 1.0: Thermophysical Database of Proteins in Solutions.

We present here a freely available web-based database, called BioMThermDB 1.0, of thermophysical and dynamic properties of various proteins and their aqueous solutions. It contains the hydrodynamic radius, electrophoretic mobility, zeta potential, self-diffusion coefficient, solution viscosity, and cloud-point temperature, as well as the conditions for those determinations and details of the experimental method. It can facilitate the meta-analysis and visualization of data, can enable comparisons, and may be useful for comparing theoretical model predictions with experiments.

A new fibrillization mechanism of ß-lactoglobulin in glycine solutions.

Even though amyloid aggregates were discovered many years ago the mechanism of their formation is still a mystery. Because of their connection to many of untreatable neurodegenerative diseases the motivation for finding a common aggregation path is high. We report a new high heat induced fibrillization path of a model protein ß-lactoglobulin (BLG) when incubated in glycine instead of water at pH 2. By combining atomic force microscopy (AFM), transmission emission microscopy (TEM), dynamic light scattering (DLS) and circular dichroism (CD) we predict that the basic building blocks of fibrils made in glycine are not peptides, but rather spheroid oligomers of different height that form by stacking of ring-like structures. Spheroid oligomers linearly align to form fibrils by opening up and combining. We suspect that glycine acts as an hydrolysation inhibitor which consequently promotes a different fibrillization path. By combining the known data on fibrillization in water with our experimental conclusions we come up with a new fibrillization scheme for BLG. We show that by changing the fibrillization conditions just by small changes in buffer composition can dramatically change the aggregation pathway and the effect of buffer shouldn't be neglected. Fibrils seen in our study are also gaining more and more attention because of their pore-like structure and a possible cytotoxic mechanism by forming pernicious ion-channels. By preparing them in a simple model system as BLG we opened a new way to study their formation.

Influence of Low Molecular Weight Salts on the Viscosity of Aqueous-Buffer Bovine Serum Albumin Solutions.

Pharmaceutical design of protein formulations aims at maximum efficiency (protein concentration) and minimum viscosity. Therefore, it is important to know the nature of protein-protein interactions and their influence on viscosity. In this work, we investigated the dependence of the viscosity of BSA in an aqueous 20 mM acetate buffer at pH = 4.3 on protein concentration and on temperature (5-45 °C). The viscosity of the solution increased with protein concentration and was 230% higher than the viscosity of the protein-free formulation at 160 mg/mL. The viscosity decreased by almost 60% in the temperature range from 5 to 45 °C. The agreement of the modified Arrhenius theory with experiment was quantitative, whereas a hard-sphere model provided only a qualitative description of the experimental results. We also investigated the viscosity of a 100 mg/mL BSA solution as a function of the concentration of added low molecular weight salts (LiCl, NaCl, KCl, RbCl, CsCl, NaBr, NaI) in the range of salt concentrations up to 1.75 mol/L. In addition, the particle size and zeta potential of BSA-salt mixtures were determined for solutions containing 0.5 mol/L salt. The trends with respect to the different anions followed a direct Hofmeister series (Cl- > Br- > I-), whereas for cations in the case of viscosity the indirect Hofmeister series was observed (Li+ > Na+ > K+ > Rb+ > Cs+), but the values of particle sizes and zeta potential did not show cation-specific effects. Since the protein is positively charged at pH = 4.3, anions are more attracted to the protein surface and shield its charge, while the interaction with cations is less pronounced. We hypothesize that salt surface charge shielding reduces protein colloidal stability and promotes protein aggregate formation.

The influence of excipients on the viscosity of monoclonal antibody solutions.

The aggregation propensity of monoclonal antibodies can be modified by adding different cosolutes into the solution. A simple coarse-grained model in the combination with the thermodynamic perturbation theory was used to predict cluster distribution and viscosity of the solutions of IgG4 monoclonal anibody in the presence of L-Arginine Hydrochloride. The data were analysed using binding polynomial to describe the binding of cosolute (Arginine) to the antibody molecule. The results show that by binding to the antibody molecule the cosolute occupies some of the binding sites of the antibody, and in this way reduces the amount of binding sites available to other antibody molecules. The aggregation propensity of the antibody molecules is therefore reduced.

CAT-Site: Predicting Protein Binding Sites Using a Convolutional Neural Network.

Identifying binding sites on the protein surface is an important part of computer-assisted drug design processes. Reliable prediction of binding sites not only assists with docking algorithms, but it can also explain the possible side-effects of a potential drug as well as its efficiency. In this work, we propose a novel workflow for predicting possible binding sites of a ligand on a protein surface. We use proteins from the PDBbind and sc-PDB databases, from which we combine available ligand information for similar proteins using all the possible ligands rather than only a special sub-selection to generalize the work of existing research. After performing protein clustering and merging of ligands of similar proteins, we use a three-dimensional convolutional neural network that takes into account the spatial structure of a protein. Lastly, we combine ligandability predictions for points on protein surfaces into joint binding sites. Analysis of our model's performance shows that its achieved sensitivity is 0.829, specificity is 0.98, and F1 score is 0.517, and that for 54% of larger and pharmacologically relevant binding sites, the distance between their real and predicted centers amounts to less than 4 Å.

Effect of Buffer on Protein Stability in Aqueous Solutions: A Simple Protein Aggregation Model.

Liquid-liquid phase separation (LLPS) of proteins has recently been associated with the onset of numerous diseases. Despite several studies in this area of protein aggregation, buffer-specific effects always seem to be overlooked. In this study we investigated the influence of buffers on the phase stability of hen egg-white lysozyme (HEWL) and its respective protein-protein interactions by measuring the cloud point temperature, second virial coefficient, and interaction diffusion coefficient of several HEWL-buffer solutions (MOPS, phosphate, HEPES, cacodylate) at pH 7.0. The results indicate that the buffer molecules, depending on their hydration, adsorb on the protein surface, and modulate their electrostatic stability. The obtained information was used to extend the recently developed coarse-grained protein model to incorporate buffer-specific effects. Treated by Wertheim's perturbation theory the model qualitatively correctly predicted the experimentally observed phase separation of all investigated HEWL-buffer solutions, and further allowed us to predict the phase stability of protein formulations even in experimentally unattainable conditions. Since the theory can be straightforwardly extended to include multiple components it presents a useful tool to study protein aggregation in crowded cell-like systems.

Studying the mechanism of phase separation in aqueous solutions of globular proteins via molecular dynamics computer simulations.

Proteins are the most abundant biomacromolecules in living cells, where they perform vital roles in virtually every biological process. To maintain their function, proteins need to remain in a stable (native) state. Inter- and intramolecular interactions in aqueous protein solutions govern the fate of proteins, as they can provoke their unfolding or association into aggregates. The initial steps of protein aggregation are difficult to capture experimentally, therefore we used molecular dynamics simulations in this study. We investigated the initial phase of aggregation of two different lysozymes, hen egg-white (HEWL) and T4 WT* lysozyme and also human lens γ-D crystallin by using atomistic simulations. We monitored the phase stability of their aqueous solutions by calculating time-dependent density fluctuations. We found that all proteins remained in their compact form despite aggregation. With an extensive analysis of intermolecular residue-residue interactions we discovered that arginine is of paramount importance in the initial stage of aggregation of HEWL and γ-D crystallin, meanwhile lysine was found to be the most involved amino acid in forming initial contacts between T4 WT* molecules.

The Role of Buffers in Wild-Type HEWL Amyloid Fibril Formation Mechanism.

Amyloid fibrils, highly ordered protein aggregates, play an important role in the onset of several neurological disorders. Many studies have assessed amyloid fibril formation under specific solution conditions, but they all lack an important phenomena in biological solutions-buffer specific effects. We have focused on the formation of hen egg-white lysozyme (HEWL) fibrils in aqueous solutions of different buffers in both acidic and basic pH range. By means of UV-Vis spectroscopy, fluorescence measurements and CD spectroscopy, we have managed to show that fibrillization of HEWL is affected by buffer identity (glycine, TRIS, phosphate, KCl-HCl, cacodylate, HEPES, acetate), solution pH, sample incubation (agitated vs. static) and added excipients (NaCl and PEG). HEWL only forms amyloid fibrils at pH = 2.0 under agitated conditions in glycine and KCl-HCl buffers of high enough ionic strength. Phosphate buffer on the other hand stabilizes the HEWL molecules. Similar stabilization effect was achieved by addition of PEG12000 molecules to the solution.

Replica Ornstein-Zernike Theory Applied for Studying the Equilibrium Distribution of Electrolytes across Model Membranes.

By means of replica Ornstein-Zernike theory (supplemented in a few cases by Monte Carlo simulations) we examined the distribution of an annealed primitive model +1:-1 electrolyte in a mixture with uncharged hard spheres, or another model +1:-1 or +2:-1 electrolyte inside and outside the quenched vesicles, decorated by a model membrane, and across the membrane phase. We explored the influence of the size and charge of the annealed fluid on the partition equilibrium, as well as the effect of the vesicle size and membrane interaction parameters (repulsive barrier height, attractive depth, and membrane width). A hydrophobic cation, present in the mixture with NaCl, slightly enhanced the concentration of sodium ions inside the model vesicle, compared to pure NaCl solution. The replica theory was in good agreement with computer simulations and as such adequate for studying partitioning of small and hydrophobic ions or hydrophobic solutes across model membranes.

DNA-Polyelectrolyte Complexation Study: The Effect of Polyion Charge Density and Chemical Nature of the Counterions.

Complexes of polycations and DNA, also known as polyplexes, have been extensively studied in the past decade, as potential gene delivery systems. Their stability depends strongly on the characteristics of the polycations, as well as the nature of the added salt. We present here a study of the DNA ionene complexation in which we used fluorescence, UV, and CD spectroscopy, combined with molecular dynamics computer simuations, to systematically examine the influence of the polycation charge density, as well as the influence of the nature of the counterion, on the stability of these systems. Ionenes as polycations, depending on their structural characteristics, have previously been found to possess low cytotoxicity, and are therefore particularly interesting as potential gene delivery agents. The results show that the DNA solutions in the presence of the polycation are more stable in the case of very large or very small ionene charge density, suggesting different mechanism of complexation. The computer simulations show that the ionenes with high charge density bind to the minor groove of the DNA molecules, while the ionenes with lower charge density bind to the major groove of the DNA. The nature of the counterions play only a minor role: precipitation of the DNA molecules occurs at slightly lower ionene concentration when fluoride counterion are present, compared to the bromide counterions.

Modelling the Correlation Between Molecular Electrostatic Potential and pKa on Sets of Carboxylic Acids, Phenols and Anilines.

Calculations of molecular electrostatic potential were correlated with experimental pKa values for different sets of acidic molecules (carboxylic acids, phenols, and anilines) to obtain linear relationships of variable quality. A single tri-parameter model function was constructed to describe the pKa dependence on MEP maxima together with two automatically generated molecular descriptors, namely the counts of carboxylic acid and amine functional groups.

How Water's Properties Are Encoded in Its Molecular Structure and Energies.

How are water's material properties encoded within the structure of the water molecule? This is pertinent to understanding Earth's living systems, its materials, its geochemistry and geophysics, and a broad spectrum of its industrial chemistry. Water has distinctive liquid and solid properties: It is highly cohesive. It has volumetric anomalies-water's solid (ice) floats on its liquid; pressure can melt the solid rather than freezing the liquid; heating can shrink the liquid. It has more solid phases than other materials. Its supercooled liquid has divergent thermodynamic response functions. Its glassy state is neither fragile nor strong. Its component ions-hydroxide and protons-diffuse much faster than other ions. Aqueous solvation of ions or oils entails large entropies and heat capacities. We review how these properties are encoded within water's molecular structure and energies, as understood from theories, simulations, and experiments. Like simpler liquids, water molecules are nearly spherical and interact with each other through van der Waals forces. Unlike simpler liquids, water's orientation-dependent hydrogen bonding leads to open tetrahedral cage-like structuring that contributes to its remarkable volumetric and thermal properties.

Volumetric and calorimetric properties of aqueous ionene solutions.

The volumetric (partial and apparent molar volumes) and calorimetric properties (apparent heat capacities) of aqueous cationic polyelectrolyte solutions - ionenes - were studied using the oscillating tube densitometer and differential scanning calorimeter. The polyion's charge density and the counterion properties were considered as variables. The special attention was put to evaluate the contribution of electrostatic and hydrophobic effects to the properties studied. The contribution of the CH2 group of the polyion's backbone to molar volumes and heat capacities was estimated. Synergistic effect between polyion and counterions was found.