Cationic Patches Research Articles

Respiratory viruses interacting with cells: the importance of electrostatics.

The COVID-19 pandemic has rekindled interest in the molecular mechanisms involved in the early steps of infection of cells by viruses. Compared to SARS-CoV-1 which only caused a relatively small albeit deadly outbreak, SARS-CoV-2 has led to fulminant spread and a full-scale pandemic characterized by efficient virus transmission worldwide within a very short time. Moreover, the mutations the virus acquired over the many months of virus transmission, particularly those seen in the Omicron variant, have turned out to result in an even more transmissible virus. Here, we focus on the early events of virus infection of cells. We review evidence that the first decisive step in this process is the electrostatic interaction of the spike protein with heparan sulfate chains present on the surface of target cells: Patches of cationic amino acids located on the surface of the spike protein can interact intimately with the negatively charged heparan sulfate chains, which results in the binding of the virion to the cell surface. In a second step, the specific interaction of the receptor binding domain (RBD) within the spike with the angiotensin-converting enzyme 2 (ACE2) receptor leads to the uptake of bound virions into the cell. We show that these events can be expressed as a semi-quantitative model by calculating the surface potential of different spike proteins using the Adaptive Poison-Boltzmann-Solver (APBS). This software allows visualization of the positive surface potential caused by the cationic patches, which increased markedly from the original Wuhan strain of SARS-CoV-2 to the Omicron variant. The surface potential thus enhanced leads to a much stronger binding of the Omicron variant as compared to the original wild-type virus. At the same time, data taken from the literature demonstrate that the interaction of the RBD of the spike protein with the ACE2 receptor remains constant within the limits of error. Finally, we briefly digress to other viruses and show the usefulness of these electrostatic processes and calculations for cell-virus interactions more generally.

Trifaceted Mickey Mouse Amphiphiles for Programmable Self-Assembly, DNA Complexation and Organ-Selective Gene Delivery.

Instilling segregated cationic and lipophilic domains with an angular disposition in a trehalose‐based trifaceted macrocyclic scaffold allows engineering patchy molecular nanoparticles leveraging directional interactions that emulate those controlling self‐assembling processes in viral capsids. The resulting trilobular amphiphilic derivatives, featuring a Mickey Mouse architecture, can electrostatically interact with plasmid DNA (pDNA) and further engage in hydrophobic contacts to promote condensation into transfectious nanocomplexes. Notably, the topology and internal structure of the cyclooligosaccharide/pDNA co‐assemblies can be molded by fine‐tuning the valency and characteristics of the cationic and lipophilic patches, which strongly impacts the transfection efficacy in vitro and in vivo. Outstanding organ selectivities can then be programmed with no need of incorporating a biorecognizable motif in the formulation. The results provide a versatile strategy for the construction of fully synthetic and perfectly monodisperse nonviral gene delivery systems uniquely suited for optimization schemes by making cyclooligosaccharide patchiness the focus.

Experimental comparison of surface chemistries for biomolecule immobilization on paper-based microfluidic devices

Biomolecules (e.g. proteins and nucleic acids) as target analytes of microfluidic paper-based analytical devices (μPADs) are usually immobilized on a cellulose paper substrate (with intrinsically anionic surface) through physical adsorptions by van der Waals forces and electrostatic interaction thanks to cationic patches on the biomolecule. However, the physical adsorption could lead to weak biomolecule-substrate binding strength and thus low biosensing performance. Benefitting from the abundance of hydroxyl groups on the cellulose paper, chemical modification based on specific surface chemistries is capable of biofunctionalization on the μPADs by providing functional groups for covalent bindings with the target biomolecule. There are many previous reports on chemical modifications of cellulose surface for improvement of biomolecule immobilization. Nevertheless, no study has been performed on experimental evaluation of modification efficiencies of various biofunctionalization methods in the context of biosensing applications. In this paper, we compare five surface chemistries for protein immobilization on μPADs made from pure cellulose paper. For each chemical modification method, surface analyses were first conducted to monitor the surface modification process. Then, paper-based fluorometric experiments and colorimetric enzyme-linked immunosorbent assays (ELISA) were carried out on paper substrates modified by the five surface chemistries to compare their efficiencies of covalent protein immobilization. Finally, a stability experiment was carried out on the five types of surface-modified paper after 30 d storage. It was demonstrated that the potassium periodate (KIO4)-modified cellulose paper has the best performance with 53% increase in the signal output and 59% decrease in background noise of the colorimetric ELISA, and only 13% bioactivity loss after the 30 d storage. The comparison results provide a valuable experimental guideline for selecting the suitable surface chemistry for protein immobilization on μPADs.

Selective Adhesive Cell Capture without Molecular Specificity: New Surfaces Exploiting Nanoscopic Polycationic Features as Discrete Adhesive Units.

This work explored how molecularly non-specific polycationic nanoscale features on a collecting surface control kinetic and selectivity aspects of mammalian cell capture. Key principles for selective collector design were demonstrated by comparing the capture of two closely related breast cancer cell lines: MCF-7 and TMX2-28. TMX2-28 is a tamoxifen-selected clone of MCF-7. The collector was a silica surface, negatively-charged at pH 7.4, containing isolated molecules (~ 8 nm diameter) of the cationic polymer, poly(dimethyl-aminoethylmethacrylate), pDMAEMA. Important in this work is the non-selective nature of the pDMAEMA interactions with cells: pDMAEMA generally adheres negatively charged particles and cells in solution. We show here that selectivity towards cells results from collector design: this includes competition between repulsive interactions involving the negative silica and attractions to the immobilized pDMAEMA molecules, the random pDMAEMA arrangement on the surface, and the concentration of positive charge in the vicinity of the adsorbed pDMAEMA chains. The latter act as nanoscopic cationic surface patches, each weakly attracted to negatively-charged cells. Collecting surfaces engineered with an appropriate amount pDMAEMA, exposed to mixtures of MCF-7 and TMX2-28 cells preferentially captured TMX2-28 with a selectivity of 2.5. (This means that the ratio of TMX2-28 to MCF cells on the surface was 2.5 times their compositional ratio in free solution.) The ionic strength-dependence of cell capture was shown to be similar to that of silica microparticles on the same surfaces. This suggests that the mechanism of selective cell capture involves nanoscopic differences in the contact areas of the cells with the collector, allowing discrimination of closely related cell line-based small scale features of the cell surface. This work demonstrated that even without molecular specificity, selectivity for physical cell attributes produces adhesive discrimination.

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways.

Mixed Phosphoinositide/Lipid Langmuir Films in the Presence of Bivalent Cations

Local accumulation of phosphoinositides (domain formation, phosphoinositide gradients) have been linked to a broad range of physiological processes. The interaction of phosphoinositides (PIPs) with bivalent cations, cationic peptides or cationic patches in proteins has been shown to promote formation of PIP enriched domains. Our study aims to investigate how the cation/PIP interaction is modulated by the presence of lipids capable of forming hydrogen bonds with PIPs, namely phosphatidylethanolamine (PE), phosphatidylinositol (PI) or cholesterol (chol).We have investigated mixed phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)/PE monolayers at the air/water interface (Langmuir films) in the presence and absence of calcium. Both 1:1 and 4:1 mixtures of PE:PI(4,5)P2 in the absence of calcium lead to an expansion of the monolayer while the addition of relatively small amounts of calcium lead to a significant condensation. While for DOPC/PI(4,5)P2 monolayers epifluorescence microscopy revealed domain formation at low surface pressures in the presence of Ca2+, DOPE/PI(4,5)P2 monolayer did not exhibit domains even in the presence of Ca2+. These data in combination with previously published results (Graber et al. Chem. Phys. Lipids 165 (2012) 696) suggest that PE in the absence of Ca2+ inserts between PI(4,5)P2 and forms hydrogen bonds with the PI(4,5)P2 headgroup. The addition of Ca2+ to this PI(4,5)P2/PE complex results in a further condensation, presumably due to a Ca2+ bridging of the PI(4,5)P2 headgroups. These results will be compared to those obtained for PE/PI and PI/PI(4,5)P2 monolayers.

Forming two-dimensional structure of DNA-functionalized Au nanoparticles via lipid diffusion in supported lipid bilayers

Two-dimensional (2D) form of DNA-functionalized gold nanoparticles (DNA–AuNPs) has been studied. To confine the DNA–AuNPs in 2D space, we utilized the strong affinity between DNA molecules and supported lipid bilayers (SLBs). DNA–AuNPs adsorbed onto cationic SLB consisting of Dimethyl dioctadecyl ammonium bromide (DDAB) on mica substrate with high affinity. Owing to the high mobility of lipid molecules, 2D diffusion and transportation of DNA–AuNPs on the SLB was achieved. Using AFM imaging we confirmed a selective adsorption of DNA–AuNPs onto cationic lipid bilayer patches on mica surface, and then we confirmed a drastic formation change of DNA–AuNPs on the SLB with annealing. We observed that the DNA–AuNPs assembled into a densely-packed single-layered 2D superstructure through DNA hybridization.

A Paradigm Change: Efficient Transfection of Human Leukemia Cells by Stimuli-Responsive Multicompartment Micelles

The controlled nonviral delivery of genetic material using cationic polymers into cells has been of interest during the past three decades, yet the ideal delivery agent featuring utmost transfection efficiency and low cytotoxicity still has to be developed. Here, we demonstrate that multicompartment micelles from stimuli-responsive triblock terpolymers, polybutadiene-block-poly(methacrylic acid)-block-poly(2-(dimethylamino)ethyl methacrylate) (BMAAD), are promising candidates. The structures exhibit a patchy shell, consisting of amphiphilic (interpolyelectrolyte complexes, MAA and D) and cationic patches (excess D), generating a surface reminiscent to those of certain viruses and capable of undergoing pH-dependent changes in charge stoichiometry. After polyplex formation with plasmid DNA, superior transfection efficiencies can be reached for both adherent cells and human leukemia cells. Compared to the gold standard PEI, remarkable improvements and a number of advantages were identified for this system, including increased cellular uptake and an improved release of the genetic material, accompanied by fast and efficient endosomal escape. Furthermore, high sedimentation rates might be beneficial regarding in vitro applications.

Phosphatidylserine Binding Is Essential for Plasma Membrane Recruitment and Signaling Function of 3-Phosphoinositide-dependent Kinase-1

3-Phosphoinositide-dependent kinase-1 (PDK1) is a ubiquitously expressed serine/threonine kinase that functions downstream of phosphoinositide 3-kinase. Although binding of 3'-phosphoinositides, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, to the pleckstrin homology (PH) domain of PDK1 is known to be essential for its interaction with and activation of downstream kinases, the mechanism by which PDK1 is recruited to the plasma membrane remains controversial. Our surface plasmon resonance analysis of the PDK1 PH domain and selected mutants shows that the PH domain specifically binds phosphatidylserine using a site that is separate from the canonical phosphoinositide-binding site. Further cell studies show that this specific phosphatidylserine binding is important for the plasma membrane localization and signaling function of PDK1.

Delineation of Lipopolysaccharide (LPS)-binding Sites on Hemoglobin: FROM IN SILICO PREDICTIONS TO BIOPHYSICAL CHARACTERIZATION

Hemoglobin (Hb) functions as a frontline defense molecule during infection by hemolytic microbes. Binding to LPS induces structural changes in cell-free Hb, which activates the redox activity of the protein for the generation of microbicidal free radicals. Although the interaction between Hb and LPS has implications for innate immune defense, the precise LPS-interaction sites on Hb remain unknown. Using surface plasmon resonance, we found that both the Hb α and β subunits possess high affinity LPS-binding sites, with K(D) in the nanomolar range. In silico analysis of Hb including phospho-group binding site prediction, structure-based sequence comparison, and docking to model the protein-ligand interactions showed that Hb possesses evolutionarily conserved surface cationic patches that could function as potential LPS-binding sites. Synthetic Hb peptides harboring predicted LPS-binding sites served to validate the computational predictions. Surface plasmon resonance analysis differentiated LPS-binding peptides from non-binders. Binding of the peptides to lipid A was further substantiated by a fluorescent probe displacement assay. The LPS-binding peptides effectively neutralized the endotoxicity of LPS in vitro. Additionally, peptide B59 spanning residues 59-95 of Hbβ attached to the surface of Gram-negative bacteria as shown by flow cytometry and visualized by immunogold-labeled scanning electron microscopy. Site-directed mutagenesis of the Hb subunits further confirmed the function of the predicted residues in binding to LPS. In summary, the integration of computational predictions and biophysical characterization has enabled delineation of multiple LPS-binding hot spots on the Hb molecule.

Single Component and Selective Competitive Protein Adsorption in a Patchy Polymer Brush: Opposition between Steric Repulsions and Electrostatic Attractions

This work explores the use of "patchy" polymer brushes to control protein adsorption rates on engineered surfaces and to bind targeted species from protein mixtures with high selectivity but without invoking molecular recognition. The brushes of interest contain embedded cationic "patches" composed of isolated adsorbed poly(l-lysine) coils (PLL) that are about 10 nm in diameter and are randomly arranged on a silica substrate. Around these patches is a protein-resistant poly(ethylene glycol) (PEG) brush that is formed from the adsorption of a PLL-g-PEG graft copolymer on the remaining silica surface. Electrostatic attractions between individual cationic patches and the negative regions of approaching proteins may be energetically insufficient to bind proteins. Furthermore, protein-patch attractions are reduced by steric repulsions between proteins and the PEG brush. We show that protein adsorption, gauged by ultimate short-term coverages and by the initial protein adsorption rate, exhibits an adhesion threshold: pure PEG brushes of the architectures employed here and brushes containing sparse loadings of PLL patches do not adsorb protein. Above a critical PLL patch loading or threshold, protein adsorption proceeds, often dramatically. The PLL patch thresholds are specific to the protein of interest, allowing surfaces to be engineered to adhesively discriminate different proteins within a mixture. The separation achieved is remarkably sharp: one protein adsorbs, but the second is completely rejected from the interface. The surfaces in this study, by virtue of their well-controlled and well-characterized patchy nature, distinguish themselves from multicomponent brushes or brushes used to end-tether peptide sequences and nucleotides.

Particle Capture via Discrete Binding Elements: Systematic Variations in Binding Energy for Randomly Distributed Nanoscale Surface Features

This work examines how the binding strength of surface-immobilized "stickers" (representative of receptors or, in nonbiological systems, chemical heterogeneities) influences the adhesion between surfaces that are otherwise repulsive. The study focuses on a series of surfaces designed with fixed average adhesive energy per unit area and demonstrates quantitatively how a redistribution of the adhesive functionality into progressively larger clusters (stronger stickers) increases the probability of adhesive events. The work employs an electrostatic model system: relatively uniform, negative 1 μm silica spheres flow gently over negative silica flats. The flats contain small amounts of randomly positioned nanoscale cationic patches. The silica-silica interaction is repulsive; however, the cationic patches (present at sufficiently low levels that the overall surface charge remains substantially negative) produce local attractions. In this study, the attractions are relatively weak so that multiple patches engage to capture flowing particles. Experiments reveal an adhesion signature characteristic of a renormalized random distribution when the sticker strength is increased at an overall fixed binding strength per unit area of surface. The form of the particle capture curves are in good quantitative agreement with a simple model that assumes only a fixed adhesion energy needed for particle capture. Aside from the quantitative details that provide a simple formalism for anticipating particle adhesion, this work demonstrates how increasing the heterogeneities in the surface functionality can cause a system to go from being nonadhesive to becoming strongly adhesive. Indeed, systems containing small amounts of discretized adhesive functionality are always more adhesive than systems in which the same functionality is distributed uniformly over the surface (the mean field scenario).

Impact of Electrostatic Deposition of Anionic Polysaccharides on the Stability of Oil Droplets Coated by Lactoferrin

The purpose of this study was to investigate the effects of three anionic polysaccharides (low methoxyl pectin (LMP), high methoxyl pectin (HMP) and alginate) on the physicochemical properties and stability of lactoferrin (LF)-coated lipid droplets. LMP, HMP and alginate were shown to adsorb to the surfaces of LF-coated droplets at neutral pH, which was primarily attributed to electrostatic attraction between anionic groups on the polysaccharide molecules and cationic patches on the protein surfaces. In the absence of polysaccharide, the LF-coated droplets were highly unstable to aggregation when heated above about 60 degrees C at pH 7, presumably because thermal denaturation of the adsorbed proteins increased droplet attraction. The addition of either LMP or HMP prior to heating greatly improved the thermal stability of the emulsions, with no aggregation being observed from 30 to 90 degrees C. On the other hand, the presence of anionic polysaccharides had little effect on emulsion stability or even promoted emulsion instability when 0 to 200 mM NaCl or CaCl2 was added. This study shows that the stability of LF-coated lipid droplets can be improved by careful selection of an appropriate type and amount of anionic polysaccharide to incorporate.

The role of nano-scale heterogeneous electrostatic interactions in initial bacterial adhesion from flow: A case study with Staphylococcus aureus

This study investigated the initial adhesion of Staphylococcus aureus from flowing buffer onto modified albumin films with the objective of probing the influence of electrostatic heterogeneity on bacterial adhesion. Electrostatic heterogeneity, on the lengthscale of 10–100 nm, was incorporated into the protein film through the irreversible random deposition of small amounts of polycation coils to produce isolated positive “patches” on the otherwise negative albumin surface before exposure to bacteria, which also possess a net negative surface charge. The system was benchmarked against an appropriate analog using 1 μm silica spheres and the same cationic patches on a silica substrate. Bacterial adhesion from flow was measured with the surface oriented vertically to eliminate gravitational forces between the bacteria and collector. In both systems, a threshold in the surface density of polycation patches needed for bacterial (or silica particle) capture indicated multivalent binding: multiple polycation patches were needed to adhere the bacteria (particles). The shifting of the threshold to greater patch concentrations at lower ionic strengths confirmed that the electrostatic interaction area (zone of influence) was a key factor in modulating the interactions. The role of the contact area in this manner is important because it enables a quantitative explanation of counterintuitive bacterial adhesion onto net negative surfaces. The study further revealed a hydrodynamic crossover from a regime where flow aids bacterial adhesion to one where flow impedes adhesion. An explanation is put forth in terms of the relative hydrodynamic and surface forces.

The impact of nanoscale chemical features on micron-scale adhesion: Crossover from heterogeneity-dominated to mean-field behavior

This work explores the impact of nanoscale surface heterogeneity, small relative to the effective contact area between two surfaces, on pairwise colloid-scale interactions. Polycation-based positive patches, of order 10 nm in diameter, arranged randomly and lying flat on otherwise negative substrates, were used to create surfaces whose competing attractive and repulsive features determined the net interactions with opposing surfaces. Lab experiments and simulations of the adhesion of gently flowing dilute negative microparticles varied particle size (0.5–2 μm), ionic strength ( κ −1 = 1–12 nm) and the density of heterogeneity on the collectors. Limiting behaviors from heterogeneity-controlled at high ionic strength to mean-field-like interactions at low ionic strength are reported. When heterogeneities are important, pairwise interactions are more attractive than predicted by average surface properties (e.g. per DLVO), and an adhesion threshold, describing the minimum average density of cationic features needed for single particle capture (adhesion), depends strongly on Debye length. In the opposite limit, the threshold becomes insensitive to the Debye length, and the average surface character approximates the interactions. An analytical treatment, reduced to a simple scaling argument predicts a −1/2 power-law dependence of the adhesion threshold on Debye length and particle size. A slightly stronger particle size dependence in experiments and simulations results from hydrodynamic contributions along with slight scaling differences in electrostatic, van der Waals, and hydrodynamic forces. An analogy to biological ligands is made for the heterogeneity-dominated limit: it is discovered, for this particular system, that engagement of as few as 20–100 cationic patches dictates particle adhesion (with details depending on flow, particle size, and ionic strength), similar to reports for selectin-mediated rolling of white blood cells during the inflammatory pathway. Also discovered is a heterogeneity-dependent crossover in the effect of ionic strength on particle capture, where added salt promotes particle adhesion in most cases but stabilizes the particles when the heterogeneity becomes relatively dense.

Dynamic adhesion behavior of micrometer-scale particles flowing over patchy surfaces with nanoscale electrostatic heterogeneity

The dynamic adhesion behavior of micrometer-scale silica particles is investigated numerically for a low Reynolds number shear flow over a planar collecting wall with randomly distributed electrostatic heterogeneity at the 10-nanometer scale. The hydrodynamic forces and torques on a particle are coupled to spatially varying colloidal interactions between the particle and wall. Contact and frictional forces are included in the force and torque balances to capture particle skipping, rolling, and arrest. These dynamic adhesion signatures are consistent with experimental results and are reminiscent of motion signatures observed in cell adhesion under flowing conditions, although for the synthetic system the particle–wall interactions are controlled by colloidal forces rather than physical bonds between cells and a functionalized surface. As the fraction of the surface ( Θ) covered by the cationic patches is increased from zero, particle behavior sequentially transitions from no contact with the surface to skipping, rolling, and arrest, with the threshold patch density for adhesion ( Θ crit ) always greater than zero and in quantitative agreement with experimental results. The ionic strength of the flowing solution determines the extent of the electrostatic interactions and can be used to tune selectively the dynamic adhesion behavior by modulating two competing effects. The extent of electrostatic interactions in the plane of the wall, or electrostatic zone of influence, governs the importance of spatial fluctuations in the cationic patch density and thus determines if flowing particles contact the wall. The distance these interactions extend into solution normal to the wall determines the strength of the particle–wall attraction, which governs the transition from skipping and rolling to arrest. The influence of Θ, particle size, Debye length, and shear rate is quantified through the construction of adhesion regime diagrams, which delineate the regions in parameter space that give rise to different dynamic adhesion signatures and illustrate selective adhesion based on particle size or curvature. The results of this study are suggestive of novel ways to control particle–wall interactions using randomly distributed surface heterogeneity.

Modulation of pH Sensitivity of Surface Charge and Aggregation Stability of Protein-Coated Lipid Droplets by Chitosan Addition

The impact of a cationic polyelectrolyte on the pH sensitivity of the electrical charge and aggregation stability of protein-coated lipid droplets was examined. One percent (w/w) corn oil-in-water emulsions containing lipid droplets coated by β-lactoglobulin [0.05% (w/w) β-Lg, 10 mM acetate buffer, pH 3] were prepared in the absence (“primary” emulsions) and presence (“secondary” emulsions) of chitosan (0 to 0.05 wt%). The pH (3 to 8) of these emulsions was adjusted, and the particle charge, particle size, creaming stability, and microstructure were measured. Chitosan adsorbed to the β-Lg-coated droplets from pH 4.5 to 7.5, which was attributed to electrostatic attraction between the cationic polyelectrolyte and anionic patches on the droplet surfaces. Droplets coated by β-Lg–chitosan had better stability to flocculation than those coated by β-Lg alone around the isoelectric point of the adsorbed proteins (pH 4.5 to 5.5), which was attributed to increased electrostatic and steric repulsion between the droplets. We have shown that chitosan may be used to modulate the electrical characteristics and stability of protein-coated lipid droplets, which may be useful in the design and formation of delivery systems for use in the food, pharmaceutical, and other industries.

Influence of pH and Ionic Strength on Formation and Stability of Emulsions Containing Oil Droplets Coated by β-Lactoglobulin−Alginate Interfaces

Emulsions of 0.1 wt % corn oil-in-water containing oil droplets coated by beta-lactoglobulin (0.009 wt % beta-Lg, 5 mM phosphate buffer, pH 7.0) were prepared in the absence and presence of sodium alginate (0 or 0.004 wt %). The pH (3-7) and ionic strength (0-250 mM NaCl) of these emulsions were adjusted, and the particle charge, particle size, and creaming stability were measured. Alginate adsorbed to the beta-Lg-coated droplets from pH 3 to 6, which was attributed to electrostatic attraction between the anionic polymer and cationic patches on the droplet surfaces. Droplets coated by beta-Lg-alginate had better stability to flocculation than those coated by beta-Lg alone, especially around the isoelectric point of the adsorbed proteins and at low ionic strengths (< 100 mM NaCl). At pH 5, alginate molecules desorbed from the droplet surfaces at high salt concentrations due to weakening of the electrostatic attraction.

Characterization of β-lactoglobulin–sodium alginate interactions in aqueous solutions: A calorimetry, light scattering, electrophoretic mobility and solubility study

The influence of pH (3–7) on the properties of sodium alginate (NaA), β-lactoglobulin (β-Lg) and their mixtures in aqueous solutions was studied at 30 °C using isothermal titration calorimetry (ITC), dynamic light scattering, turbidity, ζ-potential and soluble protein measurements. The electrical charge of isolated β-Lg went from positive to negative as the pH increased from 3 to 7 with the isoelectric point being around pH 4.8. The electrical charge of isolated sodium alginate was negative at all pH values, but was appreciably lower in magnitude from pH 3 to 4 due to partial protonation of carboxyl groups (p K a≈3.5). Light scattering measurements indicated that isolated sodium alginate was completely soluble from pH 3 to 7, but isolated β-Lg formed large complexes ( d V>200 nm) that scattered light at pH 4 and 5. When β-Lg and sodium alginate were mixed together at pH 3 and 4 they formed large complexes ( d V>1000 nm) due to electrostatic attraction between the oppositely charged molecules. At pH 5, β-Lg and sodium alginate formed fairly soluble complexes due to electrostatic attraction between the anionic polysaccharide and cationic patches on the protein surface. At pH 6 and 7, β-Lg and sodium alginate did not form complexes due to electrostatic repulsion between the similarly charged molecules. The knowledge gained in this study could be used to facilitate the rational design of food ingredients and products with desirable functional properties.

Roles of Ionic Residues of the C1 Domain in Protein Kinase C-α Activation and the Origin of Phosphatidylserine Specificity

On the basis of extensive structure-function studies of protein kinase C-alpha (PKC-alpha), we have proposed an activation mechanism for conventional PKCs in which the C2 domain and the C1 domain interact sequentially with membranes (Medkova, M., and Cho, W. (1999) J. Biol. Chem. 274, 19852-19861). To further elucidate the interactions between the C1 and C2 domains during PKC activation and the origin of phosphatidylserine specificity, we mutated several charged residues in two C1 domains (C1a and C1b) of PKC-alpha. We then measured the membrane binding affinities, activities, and monolayer penetration of these mutants. Results indicate that cationic residues of the C1a domain, most notably Arg(77), interact nonspecifically with anionic phospholipids prior to the membrane penetration of hydrophobic residues. The mutation of a single aspartate (Asp(55)) in the C1a domain to Ala or Lys resulted in dramatically reduced phosphatidylserine specificity in vesicle binding, activity, and monolayer penetration. In particular, D55A showed much higher vesicle affinity, activity, and monolayer penetration power than wild type under nonactivating conditions, i.e. with phosphatidylglycerol and in the absence of Ca(2+), indicating that Asp(55) is involved in the tethering of the C1a domain to another part of PKC-alpha, which keeps it in an inactive conformation at the resting state. Based on these results, we propose a refined model for the activation of conventional PKC, in which phosphatidylserine specifically disrupts the C1a domain tethering by competing with Asp(55), which then leads to membrane penetration and diacylglycerol binding of the C1a domain and PKC activation.