Understanding the Impacts of Molecular and Macromolecular Crowding Agents on Protein-Polymer Complex Coacervates.

Complex coacervation refers to the liquid-liquid phase separation (LLPS) process occurring between charged macromolecules. The study of complex coacervation is of great interest due to its implications in the formation of membraneless organelles (MLOs) in living cells. However, the impacts of the crowded intracellular environment on the behavior and interactions of biomolecules involved in MLO formation are not fully understood. To address this knowledge gap, we investigated the effects of crowding on a model protein-polymer complex coacervate system. Specifically, we examined the influence of sucrose as a molecular crowder and polyethylene glycol (PEG) as a macromolecular crowder. Our results reveal that the presence of crowders led to the formation of larger coacervate droplets that remained stable over a 25-day period. While sucrose had a minimal effect on the physical properties of the coacervates, PEG led to the formation of coacervates with distinct characteristics, including higher density, increased protein and polymer content, and a more compact internal structure. These differences in coacervate properties can be attributed to the effects of crowders on individual macromolecules, such as the conformation of model polymers, and nonspecific interactions among model protein molecules. Moreover, our results show that sucrose and PEG have different partition behaviors: sucrose was present in both the coacervate and dilute phases, while PEG was observed to be excluded from the coacervate phase. Collectively, our findings provide insights into the understanding of crowding effects on complex coacervation, shedding light on the formation and properties of coacervates in the context of MLOs.

Role of Domain-Domain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt.

Monoclonal antibodies (mAbs) make up a major class of biotherapeutics with a wide range of clinical applications. Their physical stability can be affected by various environmental factors. For instance, an acidic pH can be encountered during different stages of the mAb manufacturing process, including purification and storage. Therefore, understanding the behavior of flexible mAb molecules in acidic solution environments will benefit the development of stable mAb products. This study used small-angle X-ray scattering (SAXS) and complementary biophysical characterization techniques to investigate the conformational flexibility and protein-protein interactions (PPI) of a model mAb molecule under near-neutral and acidic conditions. The study also characterized the interactions between Fab and Fc fragments under the same buffer conditions to identify domain-domain interactions. The results suggest that solution pH significantly influences mAb flexibility and thus could help mAbs remain physically stable by maximizing local electrostatic repulsions when mAbs become crowded in solution. Under acidic buffer conditions, both Fab and Fc contribute to the repulsive PPI observed among the full mAb at a low ionic strength. However, as ionic strength increases, hydrophobic interactions lead to the self-association of Fc fragments and, subsequently, could affect the aggregation state of the mAb.

The Assembly Mechanism and Mesoscale Architecture of Protein-Polysaccharide Complexes Formed at the Solid-liquid Interface.

Protein-polysaccharide composite materials have generated much interest due to their potential use in medical science and biotechnology. A comprehensive understanding of the assembly mechanism and the mesoscale architecture is needed for fabricating protein-polysaccharide composite materials with desired properties. In this study, complex assemblies were built on silica surfaces through a layer-by-layer (LbL) approach using bovine beta-lactoglobulin variant A (ßLgA) and pectin as model protein and polysaccharide, respectively. We demonstrated the combined use of quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR) for elucidating the assembly mechanism as well as the internal architecture of the protein-polysaccharide complexes formed at the solid-liquid interface. Our results show that ßLgA and pectin interacted with each other and formed a cohesive matrix structure at the interface consisting of intertwined pectin chains that were cross-linked by ßLgA-rich domains. Although the complexes were fabricated in an LbL fashion, the complexes appeared to be relatively homogeneous with ßLgA and pectin molecules spatially distributed within the matrix structure. Our results also demonstrate that the density of ßLgA-pectin complex assemblies increased with both the overall and local charge density of pectin molecules. Therefore, the physical properties of the protein-polysaccharide matrix structure, including density and level of hydration, can be tuned by using polysaccharides with varying charge patterns, thus promoting the development of composite materials with desired properties.

Effects of Monovalent Salt on Protein-Protein Interactions of Dilute and Concentrated Monoclonal Antibody Formulations.

In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl (CNaCl) and monoclonal antibody (mAb) concentration (CmAb) were assessed using the diffusion interaction parameter kD and second virial coefficient B22 measured from solutions with low to moderate CmAb. The effective structure factor S(q)eff measured from concentrated mAb solutions using small-angle X-ray and neutron scattering (SAXS/SANS) was also used to characterize the PPI. Our results found that the nature of net PPI changed not only with CNaCl, but also with increasing CmAb. As a result, parameters measured from dilute and concentrated mAb samples could lead to different predictions on the stability of mAb formulations. We also compared experimentally determined viscosity results with those predicted from interaction parameters, including kD and S(q)eff. The lack of a clear correlation between interaction parameters and measured viscosity values indicates that the relationship between viscosity and PPI is concentration-dependent. Collectively, the behavior of flexible mAb molecules in concentrated solutions may not be correctly predicted using models where proteins are considered to be uniform colloid particles defined by parameters derived from low CmAb.

The so-called critical condition for polyelectrolyte-colloid complex formation.

Complexes formed between oppositely charged polyelectrolytes (PE's) and either biological or abiotic colloid particles play a central role in such remarkably diverse areas as enzyme immobilization, protein purification, growth factor delivery, personal care products, food formulations and as precursors to coacervates and multilayers. Unlike PE adsorption on oppositely charged planar surfaces-also driven by electrostatics-PE-colloid complexes are often equilibrium states exhibiting reversible formation at a well-defined "critical" colloid surface charge density. We consider how the experimentally observed breadth of this transition, for three polyelectrolyte-colloid systems, is broadened-compared to theoretical expectations-due to (1) colloid (protein) charge anisotropy, (2) colloid (micelle) polydispersity, and (3) colloid (micelle) instability.

Dilution induced coacervation in polyelectrolyte-micelle and polyelectrolyte-protein systems.

"Self-suppression", the instability of complex coacervates at high concentration, is well-known for polycation-polyanion systems, but the transient nature of those complexes impedes development of a convincing model. The stable polyelectrolyte-micelle complexes of the polycation poly(diallyldimethylammonium chloride) (PDADMAC) with mixed micelles of sodium dodecyl sulfate (SDS)/Triton X-100 (TX100); and the stable complexes of PDADMAC with bovine serum albumin (BSA) can be characterized and identified as coacervate precursors. We observe liquid-liquid phase separation upon isoionic dilution, a common facet of self-suppression. While complex coacervation usually involves association of near-neutral inter-polymer complexes, dilution-induced coacervation (DIC) proceeds differently: for both systems studied, complex size decreases near the biphasic region: inter-macromolecular complexes with hydrodynamic radius Rh∼ 100 nm dissociate to intra-polyelectrolyte complexes with Rh≤ 30 nm. Such small complexes with ≤5 bound micelles are unlikely to be net neutral. In the polyelectrolyte-protein system, complexes are even less likely to be net neutral and the effect of dilution on size is less significant, with complex size diminishing from 50 nm to 35 nm.

Precipitate-Coacervate Transformation in Polyelectrolyte-Mixed Micelle Systems.

The polycation/anionic-nonionic mixed micelle, poly(diallyldimethylammonium chloride)-sodium dodecyl sulfate/Triton X-100 (PDADMAC-SDS/TX100), is a model polyelectrolyte-colloid system in that the micellar mole fraction of SDS (Y) controls the micelle surface charge density, thus modulating the polyelectrolyte-colloid interaction. The exquisite temperature dependence of this system provides an important additional variable, controlling both liquid-liquid (L-L) and liquid-solid (L-S) phase separation, both of which are driven by the entropy of small ion release. In order to elucidate these transitions, we applied high-precision turbidimetry (±0.1 %), isothermal titration calorimetry, and epifluorescence microscopy which demonstrates preservation of micelle structure under all conditions. The L-S region at large Y including precipitation displays a remarkable linear, inverse Y-dependence of the L-S transition temperature Ts. In sharp contrast, the critical temperature for L-L coacervation Tφ, shows nearly symmetrical effects of positive and negative deviations in Y from the point of soluble complex neutrality, which is controlled in solution by the micelle charge and the number of micelles bound per polymer chain n (Zcomplex = Zpolymer + nZmicelle). In solid-like states, n no longer signifies the number of micelles bound per polymer chain, since the proximity of micelles inverts the host-guest relationship with each micelle binding multiple PE chains. This intimate binding goes hand-in-hand with the entropy of release of micelle-localized charge-compensating ions whose concentration depends on Y. These ions need not be released in L-L coacervation, but during L-S transition their displacement by PE accounts for the inverse dependence of Ts on micelle charge, Y.

Sugar-coated proteins: the importance of degree of polymerisation of oligo-galacturonic acid on protein binding and aggregation.

We have simplified the structural heterogeneity of protein-polysaccharide binding by investigating protein binding to oligosaccharides. The interactions between bovine beta-lactoglobulin A (ßLgA) and oligo-galacturonic acids (OGAs) with various numbers of sugar residues have been investigated with a range of biophysical techniques. We show that the ßLgA-OGA interaction is critically dependent on the length of the oligosaccharide. Isothermal titration calorimetry results suggest that a minimum length of 7 or 8 sugar residues is required in order to exhibit appreciable exothermic interactions with ßLgA - shorter oligosaccharides show no enthalpic interactions at any concentration ratio. When titrating ßLgA into OGAs with more than 7-8 sugar residues the sample solution also became turbid with increasing amounts of ßLgA, indicating the formation of macroscopic assemblies. Circular dichroism, thioflavin T fluorescence and small angle X-ray/neutron scattering experiments revealed two structural regimes during the titration. When OGAs were in excess, ßLgA formed discrete assemblies upon OGA binding, and no subsequent aggregation was observed. However, when ßLgA was present in excess, multi-scale structures were formed and this eventually led to the separation of the solution into two liquid-phases.

Structural mechanism of complex assemblies: characterisation of beta-lactoglobulin and pectin interactions.

Knowledge of how proteins and polysaccharides interact is the key to understanding encapsulation and emulsification in these composite systems and ultimately to understanding the structures of many biological network systems. As a model system we have studied ß-lactoglobulin A (ßLgA) interacting with pectins of various amounts and distribution patterns of charge. The studies were conducted at pH 4 at minimal ionic strength, where the ßLgA and the pectins are oppositely charged, resulting in an electrostatic attraction to each other. Isothermal titration calorimetry (ITC) experiments were performed to determine the thermodynamics associated with ßLgA-pectin titration. It was found that ßLgA only interacted with pectins with an adequate amount of charge, and that the complexation between ßLgA and pectin was a two-step process initially involving binding of the protein to available sites on the pectin, and subsequently binding of the protein onto the bound protein that has previously adsorbed. Circular dichroism (CD) and intrinsic tryptophan fluorescence were also measured of ßLgA during its interaction with the pectin samples, and show that the binding leads to significant conformational changes in ßLgA. An increase in the turbidity of the solution of the resultant complexes indicates the formation of large-scale interpolymer associations of the primary complexes mediated by protein-rich domains.