Protein Surface Coverage Research Articles

Bio-based Pickering emulsifier from mangosteen residues-derived sodium caseinate grafted spherical cellulose nanocrystals: Stability, rheological properties and microstructure studies

This study focuses on the preparation of mangosteen rind-derived nanocellulose via green ascorbic acid hydrolysis. Subsequently, milk protein-grafted nanocellulose particles were developed as a renewable Pickering emulsifier for water-oil stabilization. The stabilizing efficiency of modified nanocellulose (NC-S) at different caseinate (milk protein) concentrations (1.5, 3.0, and 4.0 % w/v) was tested in a water-in-oil emulsion (W/O ratio of 40:60). At a concentration 3.0 % w/v of caseinate (3.0NC-S), the emulsion exhibited a stronger network of adsorption between water, Pickering emulsifier, and oil. This resulted in reduced oil droplet flocculation, increased stability over a longer period, and favorable emulsifying properties, as depicted in the creaming index profile, oil droplet distribution, and rheology analysis. Since 3.0NC-S demonstrated the best colloidal stability, further focus will be placed on its microstructural properties, comparing them with those of mangosteen rind (MG), cellulose, and nanocellulose (NC-L). The XRD profile indicated that both NC-L and NC-S possessed a cellulose nanocrystal structure characterized as type I beta with a high crystallinity index above 60 %. Morphology investigation shown that the NC-L present in the spherical shape of particles with nanosized ranging at diameters of 11.27 ± 0.50 nm and length 11.76 ± 0.46 nm, while modified NC-S showed increase sized at 14.26 ± 4.60 nm and length 14.96 ± 4.94 nm. The increment of particle sizes from NC-L to NC-S indicated 2.82 × 10−15 mg/m2 of surface protein coverage by caseinate functional groups.

Modularized viromimetic polymer nanoparticle vaccines (VPNVaxs) to elicit durable and effective humoral immune responses.

Virus-like particle (VLP) vaccines had shown great potential during the COVID-19 pandemic, and was thought to be the next generation of antiviral vaccine technology due to viromimetic structures. However, the time-consuming and complicated processes in establishing a current recombinant-protein-based VLP vaccine has limited its quick launch to the out-bursting pandemic. To simplify and optimize VLP vaccine design, we herein report a kind of viromimetic polymer nanoparticle vaccine (VPNVax), with subunit receptor-binding domain (RBD) proteins conjugated to the surface of polyethylene glycol-b-polylactic acid (PEG-b-PLA) nanoparticles for vaccination against SARS-CoV-2. The preparation of VPNVax based on synthetic polymer particle and chemical post-conjugation makes it possible to rapidly replace the antigens and construct matched vaccines at the emergence of different viruses. Using this modular preparation system, we identified that VPNVax with surface protein coverage of 20%-25% had the best immunostimulatory activity, which could keep high levels of specific antibody titers over 5 months and induce virus neutralizing activity when combined with an aluminum adjuvant. Moreover, the polymer nano-vectors could be armed with more immune-adjuvant functions by loading immunostimulant agents or chemical chirality design. This VPNVax platform provides a novel kind of rapidly producing and efficient vaccine against different variants of SARS-CoV-2 as well as other viral pandemics.

Tripeptide Self-Assembled Monolayers as Biocompatible Surfaces for Cytochrome c Electrochemistry.

Biocompatible tripeptide self-assembled monolayers (SAMs) are designed with a carboxylate group on the terminal amino acid (glutamate, aspartate, or amino adipate) to electrostatically attract the lysine groups around the heme crevice in horse heart cytochrome c (cyt c), creating an electroactive protein/tripeptide/Au interfacial structure. Exposing the peptide/Au electrode to cyt c resulted in an 11 ± 3 pmol/cm2 electroactive protein surface coverage. Topographical images of the interfacial structure are obtained down to single-protein resolution by atomic force microscopy. Uniform protein monolayer assemblies are formed on the Au electrode with no major surface roughness changes. The cyt c/peptide/Au electrode systems were examined electrochemically to probe surface charge effects on the redox thermodynamics and kinetics of cyt c. Neutralization of protein surface charge due to adsorption on anionic COOH-terminated SAMs was found to change the formal potential, as determined by cyclic voltammetry. The cyt c/peptide/Au electrodes exhibit formal potentials shifted to more positive values, have a surface carboxylic acid pKa of 6 or higher, and produce effective cyt c surface charges (Zox) of -6 to -14. The Marcus theory is utilized to determine the protein electron transfer rates, which are ∼5 times faster for cyt c/tripeptide/Au compared to cyt c/11-mercaptoundecanoic acid SAMs of similar chain lengths.

Interfacial rheology of sodium caseinate/high acyl gellan gum complexes: Stabilizing oil-in-water emulsions

In this work, the effects of pH and high acyl gellan gum concentration on the adsorption kinetics and interfacial dilatational rheology of sodium caseinate/high acyl gellan gum (CN/HG) complexes were investigated using a pendant drop tensiometer. In addition, stability related properties including interfacial protein concentration, droplet charge, size, microstructure and creaming index of emulsions were studied at different HG concentration (0–0.2 wt%) and pH values (4, 5.5 and 7). The results showed that HG adsorbed onto the CN mainly through electrostatic interactions which could lead to increase the interfacial pressure (π), rates of protein diffusion (kdiff), and molecular penetration (kp). The CN/HG complexes formed thick adsorption layers around the oil droplets which significantly increased the surface dilatational modulus with the increasing HG concentration. The CN/HG complexes appeared to form more elastic interfacial films after a long-term adsorption time compared with CN alone, which could reduce the droplet coalescence and thus prevented the growth of emulsion droplets. All four phosphorylated proteins of CN (αs1-, αs2-, β-, and κ-casein) were adsorbed at the oil-water (O/W) interface as confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and surface protein coverage increased progressively with increasing HG concentration at pH 5.5, but decreased at pH 7. The CN/HG stabilized emulsions at pH 5.5 revealed the higher net charges and smaller z-average diameters than those at pH 4 and pH 7. This study provides valuable information on the use of CN/HG complexes to improve the stability and texture of food emulsions.

On the growth of the soft and hard protein corona of mesoporous silica particles with varying morphology

The characterization of the protein corona has become an essential part of understanding the biological properties of nanomaterials. This is also important in the case of mesoporous silica particles intended for use as drug delivery excipients. A combination of scattering, imaging and protein characterization techniques is used here to assess the effect of particle shape and growth of the reversible (soft) and strongly bound (hard) corona of three types mesoporous silica particles with different aspect ratios. Notable differences in the protein composition, surface coverage and particle agglomeration of the protein corona-particle complex point to specific protein adsorption profiles highly dependent on exposed facets and aspect ratio. Spherical particles form relatively homogeneous soft and hard protein coronas (approx.10 nm thick) with higher albumin content. In contrast to rod-shaped and faceted particles, which possess soft coronas weakly bound to the external surface and influenced to a greater extent by the particle morphology. These differences are likely important contributors to observed changes in biological properties, such as cell viability and immunological behaviour, with mesoporous silica particle shape.

Experimental Characterization and Mathematical Modeling of the Adsorption of Proteins and Cells on Biomimetic Hydroxyapatite.

Biomaterial development is a long process consisting of multiple stages of design and evaluation within the context of both in vitro and in vivo testing. To streamline this process, mathematical and computational modeling displays potential as a tool for rapid biomaterial characterization, enabling the prediction of optimal physicochemical parameters. In this work, a Langmuir isotherm-based model was used to describe protein and cell adhesion on a biomimetic hydroxyapatite surface, both independently and in a one-way coupled system. The results indicated that increased protein surface coverage leads to improved cell adhesion and spread, with maximal protein coverage occurring within 48 h. In addition, the Langmuir model displayed a good fit with the experimental data. Overall, computational modeling is an exciting avenue that may lead to savings in terms of time and cost during the biomaterial development process.

Behavior of molybdenum lowly doped SiO2–CaO–P2O5 composite interfacing a simulated biologic medium with serum albumin

This study is focussed on the biocompatibility evaluation by probing the affinity of bovine serum albumin (BSA) towards the surface of a silica based material containing CaMoO4 and hydroxyapatite nanocrystals. The adsorption occurring upon BSA interaction with the sample surface was investigated by Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FTIR) Spectroscopy and X-ray Photoelectron Spectroscopy (XPS). The elemental composition and evaluation of protein surface coverage based on information from the outermost few nanometres surface layer was achieved by XPS analysis. The ratio between carbon and nitrogen on the material surface after incubation in BSA solution was found to be reasonable close to the value obtained from the measurement of pure BSA, that proves the protein adsorption. The comparative analysis of FTIR spectra recorded from lyophilized protein and from biomaterial surface after protein adsorption was considered in order to obtain information about the secondary structure of the proteins. The protein surface coverage was also confirmed by SEM morphological characterization. The results obtained by in vitro test indicate that the nanostructured SiO2–CaO–P2O5−MoO3 system has a great potential for bone tissue engineering with respect to the ability to bind to serum protein.

Interaction of beta-lactoglobulin and bovine serum albumin with iron oxide (α-Fe2O3) nanoparticles in the presence and absence of pre-adsorbed phosphate

Dynamic changes in the secondary structures of BSA and β-LG observed when adsorbed onto nanoscale α-Fe2O3 surfaces. Adsorbed phosphate, a common oxyanion found in the environment, impacts protein surface coverage and modulates protein structure.

High pressure jet spray drying of condensed skim milk results in powders with enhanced interfacial properties

Abstract Condensed skim milk was processed through a continuous, custom-made high pressure jet spray dryer system (HPJ-SD) at 100–500 MPa. The resulting skim milk powders (SMPs) were evaluated for physiochemical and structural properties. The 500 MPa SMP exhibited increased particle density (1.48 g/cm3) compared to the ≤300 MPa SMP (1.33 g/cm3), but particle size analysis reflected no significant differences between these samples with D[4,3] values of 10.8 μ (100 SMP) and 12.2 μ (500 SMP). The 500 MPa SMP also had a reduced solubility and increased protein surface coverage compared to the 100 MPa SMP. The viscosity of the reconstituted HPJ-SD SMPs (9% w/w) increased from 2.3 mPa s (100 MPa) to 6.0 mPa s (500 MPa). The foam stability of the 500 MPa reconstituted SMP was exceptional with a foam head that remained stationary for 8 h (FVSI of 100%), while the foam in the 100 MPa reconstituted SMP diminished almost entirely in 2 h (FVSI of 21.3%). Evaluation of emulsification ability for reconstituted SMPs (1:9 oil in 2% diluted reconstitute w/w) revealed a 1.6-fold reduction in destabilization rate for the 500 MPa SMP compared to the 100 MPa SMP. HPJ-SD powders have physiochemical properties that can be utilized in a variety of clean label applications including ice cream or specialty beverages (e.g., cappuccino coffee).

Surface Composition and Formulation Heterogeneity of Protein Solids Produced by Spray Drying.

The aim of this study is to determine the effects of saccharide-containing excipients on the surface composition of spray-dried protein formulations and their matrix heterogeneity. Spray-dried formulations of myoglobin or bovine serum albumin (BSA) were prepared without excipient or with sucrose, trehalose, or dextrans. Samples were characterized by solid-state Fourier-transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC) and scanning electron microscopy (SEM). Protein surface coverage was determined by X-ray photoelectron spectroscopy (XPS), while conformational differences were determined by solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Structural differences were exhibited with the inclusion of different excipients, with dextran formulations indicating perturbation of secondary structure. XPS indicated sucrose and trehalose reduced protein surface concentration better than dextran-containing formulations. Using ssHDX-MS, the amount of deuterium incorporation and populations present were the largest in the samples processed with dextrans. Linear correlation was found between protein surface coverage and ssHDX-MS peak area (R2 = 0.853) for all formulations with saccharide-containing excipients. Lower molecular weight species of saccharides tend to enrich the particle surface and reduce protein concentration at the air-liquid interface, resulting in reduced population heterogeneity and improved physical stability, as identified by ssHDX-MS.

Effect of emulsifier-fat interactions and interfacial competitive adsorption of emulsifiers with proteins on fat crystallization and stability of whipped-frozen emulsions

Fat crystals and adsorbed interfacial layer are very important in controlling the stability of partly crystalline fat globules. In this study, the effects of competitive adsorption of emulsifiers, including glyceryl monooleate (GMO) and sucrose ester (SE) with milk proteins and emulsifier-fat (anhydrous milk fat, AMF; coconut oil, CO) interactions on the fat crystallization behavior and stability of whipped-frozen emulsions were investigated. Parameters such as zeta potential, surface protein coverage, rheological property, fat crystallization behavior, overrun, melting rate and microstructure of ice cream were determined. The results indicated both GMO and SE affected the crystallization behavior, changing the crystal growth, morphology and stiffness. Different mechanism was involved in fat partial coalescence in emulsion systems with different fat-emulsifier interactions. The promotion of partial coalescence in whipped-frozen emulsions with increasing concentration of GMO was mainly related to fat crystallization behavior combined with increased fat crystal growth dimension and protrusion length at the fat globule surface, since protein surface coverage was not affected by GMO concentration. However, in the case of emulsions with SE, enhancement of partial coalescence was mainly related to decreased protein surface coverage combined with less resistance of interfacial membranes to penetration by small fat crystals. Ice cream based on CO were distinguished by higher overruns and lower melting rates compared to ice cream based on AMF with the same levels of emulsifiers, especially strong fat crystal network in the presence of GMO accounts for the increased air bubble stability. These findings provides a theoretical basis for aerated food processing.

Correction to “Characterizing the Surface Coverage of Protein–Gold Nanoparticle Bioconjugates”

Correction to “Characterizing the Surface Coverage of Protein–Gold Nanoparticle Bioconjugates”

Kinetics and Mechanisms of Protein Adsorption and Conformational Change on Hematite Particles.

Adsorption kinetics and conformational changes of a model protein, bovine serum albumin (BSA, 0.1, 0.5, or 1.0 g/L), on the surface of hematite (α-Fe2O3) particles in 39 ± 9, 68 ± 9, and 103 ± 8 nm, respectively, were measured using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. As the particle size increases, the amount of adsorbed BSA decreases, but the loss in the helical structure of adsorbed BSA increases due to the stronger interaction forces between adsorbed BSA and the larger particles. On 39 or 68 nm hematite particles, refolding of adsorbed BSA can be induced by protein-protein interactions, when the protein surface coverage exceeds certain critical values. Two-dimensional correlation spectroscopy (2D-COS) analysis of time-dependent ATR-FTIR spectra indicate that the increase in the amount of adsorbed BSA occurs prior to the loss in the BSA helical structure in the initial stage of adsorption processes, whereas an opposite sequence of the changes to BSA conformation and surface coverage is observed during the subsequent refolding processes. Desorption experiments show that replacing the protein solution with water can quench the refolding, but not the unfolding, of adsorbed BSA. A kinetic model was proposed to quantitatively describe the interplay of adsorption kinetics and conformational change, as well as the effects of particle size and initial protein concentration on the rate constants of elementary steps in protein adsorption onto a mineral surface.

Cholesterol Increases Lipid Binding Rate and Changes Binding Behavior of Bacillus thuringiensis Cytolytic Protein.

Cytolytic protein (Cyt) is a member of insecticidal proteins produced by Bacillus thuringiensis. Cyt protein has activity against insect cells and mammalian cells, which differ in lipid and cholesterol composition. This study presents the lipid binding behavior of Cyt2Aa2 protein on model membranes containing different levels of cholesterol content by combining Quartz Crystal Microbalance with Dissipation (QCM-D) and Atomic Force Microscopy (AFM). QCM-D results revealed that cholesterol enhances the binding rate of Cyt2Aa2 protein onto lipid bilayers. In addition, the thicker lipid bilayer was observed for the highest cholesterol content. These results were confirmed by AFM. The analysis of protein surface coverage as a function of time showed a slower process for 5:0 and 5:0.2 (POPC:Chol) ratios than for 5:1 and 5:2 (POPC:Chol) ratios. Significantly, the Cyt2Aa2-lipid binding behavior and the protein–lipid layer were different for the 5:3 (POPC:Chol) ratio. Furthermore, AFM images revealed a transformation of Cyt2Aa2/lipid layer structure from strip pattern to ring shape structures (which showed a strong repulsion with AFM tip). In summary, cholesterol increases the binding rate and alters the lipid binding behavior of Cyt2Aa2 protein, although it is not required for Cyt2Aa2 protein binding onto lipid bilayers.

Impact of protein surface coverage and layer thickness on rehydration characteristics of milk serum protein/lactose powder particles

Spray-dried powders were produced from milk serum protein concentrate and lactose in varying ratios, and the rehydration characteristics of the powders were evaluated. The dissolution rate was estimated with a flow-cell based technique, and the external and internal distribution of the powder components were evaluated with X-ray photoelectron spectroscopy and confocal Raman microscopy, respectively. The surface of the powder particles is more or less covered by a thin protein layer. A phase segregation between protein and lactose is observed in the interior of the particle resulting in a protein rich layer in the vicinity of the surface. However, the protein layer in the vicinity of the particle surface tends to become thinner as the bulk protein concentration increases in the powders (from 10 to 60% w/w). The time for the spontaneous imbibition to occur show a linear correlation with the protein surface coverage. The dissolution rate of powders containing 0.1% w/w protein is around 60 times faster than for a powder containing 1% w/w protein but the dissolution rate of powders containing 1% and 100% w/w differ only by a factor of 2. Thus, it is suggested that the outer protein layer becomes denser at the interface as the protein content increases in the powders, thereby causing poorer rehydration characteristics of the powders (especially for low protein concentrations 0.1–1% w/w). This insight has relevance for the formulation of whey protein powders with improved rehydration characteristics.

Auto‐regulated Protein Assembly on a Supramolecular Scaffold

AbstractControlled protein assembly provides a means to regulate function. Supramolecular building blocks, including rigid macrocycles, are versatile triggers of protein assembly. Now it is shown that sulfonato‐calix[8]arene (sclx8) mediates the formation of cytochrome c tetramers in solution. This tetramer spontaneously disassembles at ≥2 equivalents of sclx8, providing a remarkable example of auto‐regulation. Using X‐ray crystallography the sclx8 binding sites on cytochrome c were characterized. Crystal structures at different protein–ligand ratios reveal varying degrees (up to 35 %) of protein surface coverage by the flexible calixarene and suggest a mechanism for oligomer disassembly. The solution structure of the oligomer was characterized by small‐angle X‐ray scattering. Overall, the data indicate calixarene‐controlled protein assembly and disassembly without the requirement for a competitive inhibitor, and point to protein encapsulation by a flexible macrocycle.

Auto-regulated Protein Assembly on a Supramolecular Scaffold.

Controlled protein assembly provides a means to regulate function. Supramolecular building blocks, including rigid macrocycles, are versatile triggers of protein assembly. Now it is shown that sulfonato-calix[8]arene (sclx8 ) mediates the formation of cytochrome c tetramers in solution. This tetramer spontaneously disassembles at ≥2 equivalents of sclx8 , providing a remarkable example of auto-regulation. Using X-ray crystallography the sclx8 binding sites on cytochrome c were characterized. Crystal structures at different protein-ligand ratios reveal varying degrees (up to 35 %) of protein surface coverage by the flexible calixarene and suggest a mechanism for oligomer disassembly. The solution structure of the oligomer was characterized by small-angle X-ray scattering. Overall, the data indicate calixarene-controlled protein assembly and disassembly without the requirement for a competitive inhibitor, and point to protein encapsulation by a flexible macrocycle.

Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates.

Functional enzyme-nanoparticle bioconjugates are increasingly important in biomedical and biotechnology applications such as drug delivery and biosensing. Optimization of the function of such bioconjugates requires careful control and characterization of their structures and activity, but current methods are inadequate for this purpose. A key shortcoming of existing approaches is the lack of an accurate method for quantitating protein content of bioconjugates for low (monolayer) surface coverages. In this study, an integrated characterization methodology for protein-gold nanoparticle (AuNP) bioconjugates is developed, with a focus on site-specific attachment and surface coverage of protein on AuNPs. Single-cysteine-containing mutants of dihydrofolate reductase are covalently attached to AuNPs with diameters of 5, 15, and 30 nm, providing a range of surface curvature. Site-specific attachment to different regions of the protein surface is investigated, including attachment to a flexible loop versus a rigid α helix. Characterization methods include SDS-PAGE, UV-vis spectrophotometry, dynamic light scattering, and a novel fluorescence-based method for accurate determination of low protein concentration on AuNPs. An accurate determination of both protein and AuNP concentration in conjugate samples allows for the calculation of the surface coverage. We find that surface coverage is related to the surface curvature of the AuNP, with a higher surface coverage observed for higher surface curvature. The combination of these characterization methods is important for understanding the functionality of protein-AuNP bioconjugates, particularly enzyme activity.

Efficient encapsulation of proteins with random copolymers

Membraneless organelles are aggregates of disordered proteins that form spontaneously to promote specific cellular functions in vivo. The possibility of synthesizing membraneless organelles out of cells will therefore enable fabrication of protein-based materials with functions inherent to biological matter. Since random copolymers contain various compositions and sequences of solvophobic and solvophilic groups, they are expected to function in nonbiological media similarly to a set of disordered proteins in membraneless organelles. Interestingly, the internal environment of these organelles has been noted to behave more like an organic solvent than like water. Therefore, an adsorbed layer of random copolymers that mimics the function of disordered proteins could, in principle, protect and enhance the proteins' enzymatic activity even in organic solvents, which are ideal when the products and/or the reactants have limited solubility in aqueous media. Here, we demonstrate via multiscale simulations that random copolymers efficiently incorporate proteins into different solvents with the potential to optimize their enzymatic activity. We investigate the key factors that govern the ability of random copolymers to encapsulate proteins, including the adsorption energy, copolymer average composition, and solvent selectivity. The adsorbed polymer chains have remarkably similar sequences, indicating that the proteins are able to select certain sequences that best reduce their exposure to the solvent. We also find that the protein surface coverage decreases when the fluctuation in the average distance between the protein adsorption sites increases. The results herein set the stage for computational design of random copolymers for stabilizing and delivering proteins across multiple media.

Impact of surface properties on morphology of spray-dried milk serum protein/lactose systems

This study investigated milk serum protein concentrate/lactose systems with varying ratios and how the morphology of the spray-dried particles of these systems could be described by the surface properties of the feed as well as the protein surface coverage of the particles. An extrapolation of the surface pressure of the feed to 0.3 s, the approximate time for molecular diffusion in an atomised droplet in the spray-dryer, showed a relationship with the particle morphology. At low protein concentrations (<1%), the particles were almost totally smooth. At higher protein concentrations (≥1%), the particles became dented and ridged, and these tended to become deeper and thicker as the protein concentration increased. It is suggested that the surface pressure of the feed at low protein concentrations is the most prominent surface property, whereas the modulus of elasticity seems to be the most prominent surface property for particle surface deformation at higher protein concentrations.