Function Of Protein Concentration Research Articles

Effective interactions in protein solutions with and without clustering

Protein interactions in solution and the question of cluster formation are of paramount importance to a wide range of research fields and industrial applications. Protein clustering can arise due to a delicate interplay between the short-range attraction and the long-range repulsion in the interaction potential. This study employs small angle X-ray scattering (SAXS) to examine the clustering behavior of three protein systems in solution: bovine serum albumin (BSA), bovine β-lactoglobulin (BLG) and lysozyme (LYZ) under varying protein concentrations, ionic strengths and temperatures. The goal is to elucidate the influence of the attractive potential’s nature on clustering behavior. BSA serves as a reference system, where electrostatic repulsions dominate the effective interactions, resulting in a mean protein–protein distance as a function of protein concentration which follows the 1/3 power law and precludes protein cluster formation. In contrast, BLG and LYZ solutions exhibit exponents less than 1/3, indicating concentration-dependent cluster formation. The effective interactions are well described using a two-Yukawa type potential comprising a long-ranged repulsion and a short-ranged attraction. While increasing ionic strength by adding NaCl reduced repulsion and correlation in all three systems, the response of protein solutions to ionic strength differed for clustering systems of BLG and LYZ. BLG solutions exhibited a reduced attraction with increasing ionic strength, leading to cluster dissolution, while LYZ solutions experienced enhanced attraction, favoring larger cluster formation. Notably, BLG solutions showed minimal temperature dependence, while LYZ solutions exhibited increased attraction with decreasing temperature, further promoting cluster formation. These results demonstrate the crucial role of short-ranged attractions of specific nature in determining protein clustering behavior. A thorough understanding of protein interactions is essential for predicting protein clustering and phase behavior.

Structural insights into acidic heating-induced amyloid fibrils derived from soy protein as a function of protein concentration

Amyloid fibrillation of food-derived dietary proteins has been increasingly accepted as an effective approach to enhance protein functionality. Soy protein, an abundant and environmentally sustainable protein, has been widely used in food applications. However, the mechanism for amyloid fibrillation at various protein concentrations during the acidic heating process is not fully understood, along with an incomplete understanding of the structural polymorphism. In this study, we systematically investigated the influence of protein concentration on amyloid fibrillation of soy protein by means of atomic force microscopy (AFM), 1-anililo-naphthalene-8-sulfonate (ANS) and thioflavin T (Th T) fluorescence, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectrometer (FTIR). AFM analysis showed that soy protein-derived amyloid fibrils (SAFs) demonstrated very rich and diverse polymorphism and exhibited at least five kinds of long-rigid fibrils that had distinct periodicity, height, and handedness. The polypeptides hydrolysis behavior and the parallel to antiparallel β-sheet ratios were found to be protein concentration-dependent. Results obtained from this study will lead to a deeper understanding of formation mechanism of SAFs and lay the foundation for SAFs severing as functional ingredients in food field.

Pressure, shear, thermal, and interaction effects on quality attributes of pea–dairy protein colloidal dispersions

This study evaluated the feasibility of producing stable protein colloidal dispersion by blending dairy and pea proteins using ultra shear technology (UST). The influence of different UST process parameters (shear, temperature, pressure, and interactions) on the quality attributes of milk and pea protein suspensions of different protein proportions viz., milk:pea 1:0.5, 1:1, and 1:3 protein ratios was evaluated. UST treatment was performed at 400 MPa pressure and process temperatures of 40 and 70 °C. High pressure processing (HPP) at 400 MPa at 25 ± 2 °C and thermal treatment at 72 °C were conducted to study pressure-only and temperature-only effects. UST treatment reduced particle size at lower protein concentrations, however, aggregation of particles was observed above certain concentrations. For example, for the UST treatment at 40 °C, the mean particle size of milk:pea 1:0.5 and 1:3 protein suspensions were 2.48 and 23.06 μm, respectively. HPP and thermal treatments did not alter the particle size of the samples. UST treatment produced stable colloidal dispersions, with no sedimentation up to 15 days storage at 4 °C. UST treatment created homogenous stable dairy–pea protein blends, emulsions, or gel structures depending on treatment intensity and protein concentration. • Ultra-shear technology produced stable milk-pea protein blends without preservatives. • UST treatment increased viscosity of liquid foods as function of protein content. • UST modified the particle size characteristic and prevented sedimentation in blends.

Graphene Oxide/Silver Nanoparticles Platforms for the Detection and Discrimination of Native and Fibrillar Lysozyme: A Combined QCM and SERS Approach.

We propose a sensing platform based on graphene oxide/silver nanoparticles arrays (GO/AgNPs) for the detection and discrimination of the native and toxic fibrillar forms of an amyloid-prone protein, lysozyme, by means of a combination of Quartz Crystal Microbalance (QCM) and Surface Enhanced Raman Scattering (SERS) measurements. The GO/AgNPs layer system was obtained by Langmuir-Blodgett assembly of the silver nanoparticles followed by controlled adsorption of GO sheets on the AgNPs array. The adsorption of native and fibrillar lysozyme was followed by means of QCM, the measurements provided the kinetics and the mechanism of adsorption as a function of protein concentration as well as the mass and thickness of the adsorbed protein on both nanoplatforms. The morphology of the protein layer was characterized by Confocal Laser Scanning Microscopy experiments on Thioflavine T-stained samples. SERS experiments performed on arrays of bare AgNPs and of GO coated AgNP after native, or fibrillar, lysozyme adsorption allowed for the discrimination of the native form and toxic fibrillar structure of lysozyme. Results from combined QCM/SERS studies indicate a general construction paradigm for an efficient sensing platform with high selectivity and low detection limit for native and amyloid lysozyme.

Assessing the chargeability and separability of oat groat particles through sieving combined with triboelectrification-based approach

• Oat groats were sieved at six different aperture sizes to produce twelve fractions. • Sieved fractions were analyzed for protein, particle size and tribo-charge behavior. • Particle diameters and protein content of sieved fractions were linearly correlated. • Sieved bran fractions were rich in protein and acquired high charges at laminar flow. • Sieved starchy fractions were enriched in protein by tribo-electrostatic separation. The possibility of fractionation of dehulled oat groat particles was assessed through combined sieving and tribo-electrostatic separation approach. Through this process, oat groats were initially sieved at six different aperture sizes of 53 to 425 μm, resulting in the production of twelve different fractions including six starch-rich fractions as well as six bran-rich fractions among which the bran-rich fractions had significantly higher protein concentration (15.5–19.6%) compared to the starch-rich fractions (9.7–14.0%). A linear relationship (R 2 = 0.9554) was found between protein content and mean particle diameters (D 50 ) of all sieved fractions. The tribo-charging behavior of all sieved fractions was assessed as a function of particle size and protein content. Bran-rich particles despite their large particle sizes were acquired higher charge-to-mass (nC/g) ratios compared to starch-rich fractions at the laminar air flow rate (Re = 2091) in contact with polytetrafluoroethylene (PTFE) tribo-charging tube. To exclusively evaluate the tribo-charging behavior of the fractions as a function of protein content, the specific charge (nC/g) values were recalculated to charge density (nC/m 2 ) values and a three-stage incremental trend was observed between charge density and protein content of the fractions at the laminar air flow rate. Starch-rich fractions exhibited totally different tribo-charging behavior at the turbulent (Re = 4182) air flow rate due to the large surface-to-volume ratio of their fine small particles that resulted in enhanced particle–wall collisions and eventually acquisition of higher specific charge values compared to their equivalent sieved bran-rich fractions. The starch-rich fraction sieved at aperture size of 106 μm with protein content of 10.1% was further fractionated through single- and multi-stage tribo-electrostatic separation processes where its protein content was sharply enhanced by ~80% equivalent to over 55% protein separation efficiency.

Monitoring pH and whey protein digestion by TD-NMR and MRI in a novel semi-dynamic in vitro gastric simulator (MR-GAS)

Gastric digestion is crucial for protein breakdown. Magnetic resonance techniques have a great deal of potential but remain underexplored with regard to their application in the study of food digestion via MRI-markers, such as transverse (R2) and longitudinal (R1) relaxation rates. R2 has been used to monitor gastric digestion of whey protein gels, but only in a static in vitro model. It is essential to investigate whether relaxation rates can be valid measures of digestion under dynamic circumstances. We developed a novel MRI-compatible semi-dynamic gastric simulator (MR-GAS) that includes controlled gastric secretion, emptying and mixing at body temperature. PH and protein hydrolysis were measured during protein gel digestion in the MR-GAS. R2 and R1 of the supernatant were measured by time-domain nuclear magnetic resonance (TD-NMR). The stomach chamber of the MR-GAS was also scanned with MRI to measure R2 and R1. For TD-NMR, 99% of the variance in R2 and 96% of variance in R1 could be explained as a function of protein concentration and [H+]. For MRI, the explained variances were 99% for R2 and 60% for R1. From these analysis, the obtained equations enabled the prediction of protein concentration and pH by R2 and R1. The normalised root mean squared deviation of the predictions for protein concentration were 0.15 (NMR) and 0.18 (MRI), and for pH were 0.12 (NMR) and 0.29 (MRI). In conclusion, the MR-GAS model may be used in a clinical MRI to monitor gastric digestion under in vitro dynamic circumstances, by measuring R2 and R1. These results underscore the potential of MRI to monitor nutrients hydrolysis and pH changes in future in vivo studies.

The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors

The maturation of coronavirus SARS-CoV-2, which is the etiological agent at the origin of the COVID-19 pandemic, requires a main protease Mpro to cleave the virus-encoded polyproteins. Despite a wealth of experimental information already available, there is wide disagreement about the Mpro monomer-dimer equilibrium dissociation constant. Since the functional unit of Mpro is a homodimer, the detailed knowledge of the thermodynamics of this equilibrium is a key piece of information for possible therapeutic intervention, with small molecules interfering with dimerization being potential broad-spectrum antiviral drug leads. In the present study, we exploit Small Angle X-ray Scattering (SAXS) to investigate the structural features of SARS-CoV-2 Mpro in solution as a function of protein concentration and temperature. A detailed thermodynamic picture of the monomer-dimer equilibrium is derived, together with the temperature-dependent value of the dissociation constant. SAXS is also used to study how the Mpro dissociation process is affected by small inhibitors selected by virtual screening. We find that these inhibitors affect dimerization and enzymatic activity to a different extent and sometimes in an opposite way, likely due to the different molecular mechanisms underlying the two processes. The Mpro residues that emerge as key to optimize both dissociation and enzymatic activity inhibition are discussed.

Homogenization and sodium hydrogen phosphate induced effect on physical and rheological properties of ultrafilterd concentrated milk.

The online version contains supplementary material available at 10.1007/s13197-021-05097-2.

Concentration-dependent changes in the reaction behavior of whey proteins: Diffusion-controlled or transition state-controlled reactions?

The denaturation of whey proteins can be used in a controlled manner to design protein-based products with new specific structures or functionalities e.g., meat and dairy analogues, as well as protein-based emulsifiers and thickeners with specific functionalities. For this, the influence of thermal and/or mechanical treatment, and milieu conditions including the protein concentration, on the denaturation reaction must be determined. Previous results of whey protein denaturation at concentrations above 50% suggested a strong change in the reaction behavior of whey proteins compared to more dilute conditions (concentration < 20%). Therefore, in this study the reaction behavior of whey proteins at a broad range of concentrations 17–70% after thermal and mechanical treatment was investigated. The results of this study suggest that the reaction limiting step depends on the concentration of the system. Calculations of the collision rate as a function of protein concentration indicate that the collision theory can only be applied to explain the denaturation reaction mechanisms for certain conditions. At dilute- and semi-dilute conditions, since the protein molecules are quasi-isolated and unentangled, the diffusion of molecules controls the aggregation step, and consequently the denaturation reaction is a diffusion-limited reaction. In contrast at concentrated conditions, the protein molecules are entangled, which can hinder the unfolding of the globular structure and consequently the formation of a transition-state becomes the limiting step.

Abstract 6571: Plasma protein-protein interactome (PPI) maps derived from the protein corona captured at the nano-bio interface of nanoparticles reveal differential networks for non-small cell lung cancer (NSCLC) and control subjects

Abstract Introduction: Understanding changes in PPI maps from a healthy and diseased state can illuminate our understanding of biological changes and disease processes. PPI maps enable a higher order of information than a simple listing of components by providing functional context, yet existing maps grossly underrepresent the total biological information potential of PPIs. Herein, we describe Proteograph, a novel platform that leverages the nano-bio interactions of nanoparticles (NPs) for deep and unbiased proteomic sampling that can provide insights on PPI across biological samples. Proteograph leverages the protein corona that forms on the surface of NPs as a function of their distinct biophysicochemical properties. NPs reproducibly bind subsets of proteins from biofluids as a function of protein concentration, protein-NP affinity, and protein-protein interactions to form a corona on the NP surface. We have employed Proteograph to quantify known PPIs using a panel of 3 distinct NPs to capture plasma proteins and derive maps of NSCLC and control subjects in order to identify biological changes in interactions, potentially indicative of health and disease. Method and Results: We collected plasma samples from 288 subjects: healthy (n=82), comorbid (n=81) and NSCLC stages I-IV (n=125). In this initial study, we used three NPs with distinct properties and evaluated the protein corona of plasma samples by mass spectrometry (MS) to quantify 1,235 protein groups (1% FDR). A fully automated assay workflow enabled preparation of 3 NPs' corona for MS analysis across 288 subjects in approximately 6 days. We mapped the protein groups to a PPI map derived from the STRING database. Partitioning the network into clusters identified 9 interaction clusters with greater than 10 protein members. These clusters enabled us to investigate differences in the PPI networks between NSCLC patients vs. controls. Evaluating the expression of proteins in these groups, we identified interaction clusters that had significant differences between cancer vs. control (t-test, p &amp;lt; 0.01 Bonferroni corrected). Six of the clusters show differential behavior between NSCLC vs. healthy controls (p &amp;lt; 0.01). Two of these clusters show differential behavior between NSCLC vs. healthy and comorbid (p &amp;lt; 0.01). Investigation of these differentially expressed clusters reveals links to known cancer biology with proteins related to the immune system and endocytosis pathways. Discussion: We have used the Proteograph platform to identify PPI clusters that are differentiated between NSCLC and control individuals. We believe the efficiency of the Proteograph platform applied to sufficiently powered studies may enable comprehensive understanding of known PPIs, and potentially infer and confirm new PPIs, in health and disease. Citation Format: Asim Siddiqui, John E. Blume, William C. Manning, Gregory Troiano, Philip Ma, Robert Langer, Vivek Farias, Omid C. Farokhzad. Plasma protein-protein interactome (PPI) maps derived from the protein corona captured at the nano-bio interface of nanoparticles reveal differential networks for non-small cell lung cancer (NSCLC) and control subjects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6571.

Abstract 2848: Analytical validation of the multi-nanoparticle Proteograph platform for rapid and deep proteomic profiling

Abstract Introduction: Plasma proteins should be useful biomarkers for disease detection, yet few proteins (~120) are FDA approved. Productive biomarker discovery studies are resource-limited due to the complex biochemical fractionation methods used to address the inherent challenges in unbiased plasma profiling such as the large dynamic range. Herein, we describe Proteograph, a novel platform that leverages the nano-bio interactions of nanoparticles (NPs) for deep and unbiased proteomic sampling. NPs reproducibly bind subsets of proteins from biofluids as a function of protein concentration, protein-NP affinity, and protein-protein interactions to form a corona on the NP surface. The corona composition is directly a function of the NPs' biophysicochemical properties and requires no prior knowledge of the proteins that might be selected by each distinctly engineered NP. With an optimized panel of 10 NPs, we can broadly and deeply interrogate the plasma proteome and rapidly quantify potential biomarkers. The highly parallel NP workflow makes large studies practicable and should improve biomarker discovery and validation. Methods and Results: We have screened 200+ NPs with different biophysicochemical properties and selected a panel of 10 based on protein detection. A fully automated assay workflow was developed that can process the 10 NP panel across 8 plasma samples in a 7 hr assay. Evaluation of this panel across 16 individual plasma samples detected 2,009 protein groups (1% protein FDR, 84% with 2 or more peptides). Accuracy was demonstrated with spike-recovery experiments using 4 NPs in which CRP, Angiogenin and S100a8/9 were added to plasma at 2X, 5X, 10X, and 100X of measured endogenous levels. Linear model fits for NP corona MS signal vs. ELISA were created with mean slopes of 1.06 ± 0.22 and mean adjusted-r2 of 0.95 ± 0.05. Precision was demonstrated across 3 NPs using three assay replicates in which the mean of the median CVs for each NP is 24%. The depth of plasma proteome coverage for the 10 NP panel using a pooled plasma sample was determined by comparison of the NP-detected proteins to published MS intensities and spanned nearly the entire reported range. Examining protein annotations (e.g., GO Cellular Compartment and Biological Process, KEGG and Pfam) within each NP corona reveals correlations by 1D-enrichment analysis between protein annotations and NP biophysicochemical properties suggesting specific relationships at the nano-bio surface. Discussion: We have demonstrated the selection and optimization of a panel of 10 NPs for plasma proteome profiling. We have also demonstrated the breadth and depth of this panel's ability to accurately and precisely quantify proteins from plasma. We believe the robustness and scalability of the Proteograph platform could enable population-scale deep and unbiased proteomics analysis previously not feasible using existing workflows. Citation Format: John E. Blume, William C. Manning, Gregory Troiano, Asim Siddiqui, Philip Ma, Robert Langer, Vivek Farias, Omid C. Farokhzad. Analytical validation of the multi-nanoparticle Proteograph platform for rapid and deep proteomic profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2848.

Kinetics of Fast Tetramerization of the Huntingtin Exon 1 Protein Probed by Concentration-Dependent On-Resonance R1ρ Measurements.

An approach for the quantitative description of the kinetics of very fast exchange processes (τex < 50-100 μs) associated with transient, reversible protein oligomerization, is presented. We show that on-resonance 15N-R1ρ measurements conducted as a function of protein concentration at several spin-lock radio frequency field strengths are indispensable for unambiguous determination of the rate constants for interconversion between monomeric and higher order oligomeric species. The approach is experimentally demonstrated on the study of fast, reversible tetramerization of the full-length Huntingtin exon 1 protein, httex1, responsible for Huntington's disease. Incorporation of concentration-dependent 15N-R2,eff data, obtained from on-resonance R1ρ measurements performed at three spin-lock field strengths, into analysis of the kinetic scheme describing reversible tetramerization of httex1 allowed us to uniquely determine the rate constants of interconversion between the various species. This approach serves as a valuable complement to the existing array of NMR techniques for studying early, transient oligomerization events in protein aggregation pathways.

Effect of Protein Corona on The Transfection Efficiency of Lipid-Coated Graphene Oxide-Based Cell Transfection Reagents.

Coating graphene oxide nanoflakes with cationic lipids leads to highly homogeneous nanoparticles (GOCL NPs) with optimised physicochemical properties for gene delivery applications. In view of in vivo applications, here we use dynamic light scattering, micro-electrophoresis and one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis to explore the bionano interactions between GOCL/DNA complexes (hereafter referred to as ”grapholipoplexes”) and human plasma. When exposed to increasing protein concentrations, grapholipoplexes get covered by a protein corona that evolves with protein concentration, leading to biocoronated complexes with modified physicochemical properties. Here, we show that the formation of a protein corona dramatically changes the interactions of grapholipoplexes with four cancer cell lines: two breast cancer cell lines (MDA-MB and MCF-7 cells), a malignant glioma cell line (U-87 MG) and an epithelial colorectal adenocarcinoma cell line (CACO-2). Luciferase assay clearly indicates a monotonous reduction of the transfection efficiency of biocoronated grapholipoplexes as a function of protein concentration. Finally, we report evidence that a protein corona formed at high protein concentrations (as those present in in vivo studies) promotes a higher capture of biocoronated grapholipoplexes within degradative intracellular compartments (e.g., lysosomes), with respect to their pristine counterparts. On the other hand, coronas formed at low protein concentrations (human plasma = 2.5%) lead to high transfection efficiency with no appreciable cytotoxicity. We conclude with a critical assessment of relevant perspectives for the development of novel biocoronated gene delivery systems.

Rotational and Translational Diffusion of Proteins as a Function of Concentration.

Atomistic simulations of three different proteins at different concentrations are performed to obtain insight into protein mobility as a function of protein concentration. We report on simulations of proteins from diluted to the physiological water concentration (about 70% of the mass). First, the viscosity was computed and found to increase by a factor of 7–9 going from pure water to the highest protein concentration, in excellent agreement with in vivo nuclear magnetic resonance results. At a physiological concentration of proteins, the translational diffusion is found to be slowed down to about 30% of the in vitro values. The slow-down of diffusion found here using atomistic models is slightly more than that of a hard sphere model that neglects the electrostatic interactions. Interestingly, rotational diffusion of proteins is slowed down somewhat more (by about 80–95% compared to in vitro values) than translational diffusion, in line with experimental findings and consistent with the increased viscosity. The finding that rotation is retarded more than translation is attributed to solvent-separated clustering. No direct interactions between the proteins are found, and the clustering can likely be attributed to dispersion interactions that are stronger between proteins than between protein and water. Based on these simulations, we can also conclude that the internal dynamics of the proteins in our study are affected only marginally under crowding conditions, and the proteins become somewhat more stable at higher concentrations. Simulations were performed using a force field that was tuned for dealing with crowding conditions by strengthening the protein–water interactions. This force field seems to lead to a reproducible partial unfolding of an α-helix in one of the proteins, an effect that was not observed in the unmodified force field.

Power ultrasound as a tool to improve the processability of protein-enriched fermented milk gels for Greek yogurt manufacture

One approach to avoid production of acid whey during the manufacture of high-protein yogurt and related products is to concentrate the milk before fermentation. However, the resultant gels are firm so that stirring in the tank and further processing are difficult on an industrial scale. We hypothesize that power ultrasound (US) during fermentation softens the gel because sound waves cause cavitation and strong shear forces in the fluid. Skim milk was standardized to different protein contents up to 12%, heated (85°C, 30 min), and acidified with thermophilic or mesophilic starter cultures. An excessive increase in gel firmness as a function of protein content was detected. In the next series of experiments, US was applied during fermentation. Milks (10% protein) were acidified at 43.5°C and sonicated from pH 5.8 to 5.1 with a sonotrode (20 kHz, 20 W). Immediately after fermentation, gels were agitated using a rheometer with a vane geometry. The maximum torque required to break the gel was reduced by 75% following US, and gel firmness was reduced by 80%. Gels were then processed into stirred yogurt and analyzed. Sonicated samples were smoother with fewer large aggregates. Confocal laser scanning microscopy images suggested a less cohesive structure and more compact microgel particles, resulting in reduced viscosity. We concluded that US is a promising tool to weaken the gel and facilitate further processing. This enables new approaches for the manufacture of Greek yogurt, particularly in regard to avoiding production of acid whey and developing products with novel textures.

Characterization of a Surface-Active Protein Extracted from a Marine Strain of Penicillium chrysogenum.

Marine microorganisms represent a reservoir of new promising secondary metabolites. Surface-active proteins with good emulsification activity can be isolated from fungal species that inhabit the marine environment and can be promising candidates for different biotechnological applications. In this study a novel surface-active protein, named Sap-Pc, was purified from a marine strain of Penicillium chrysogenum. The effect of salt concentration and temperature on protein production was analyzed, and a purification method was set up. The purified protein, identified as Pc13g06930, was annotated as a hypothetical protein. It was able to form emulsions, which were stable for at least one month, with an emulsification index comparable to that of other known surface-active proteins. The surface tension reduction was analyzed as function of protein concentration and a critical micellar concentration of 2 μM was determined. At neutral or alkaline pH, secondary structure changes were monitored over time, concurrently with the appearance of protein precipitation. Formation of amyloid-like fibrils of SAP-Pc was demonstrated by spectroscopic and microscopic analyses. Moreover, the effect of protein concentration, a parameter affecting kinetics of fibril formation, was investigated and an on-pathway involvement of micellar aggregates during the fibril formation process was suggested.

TD‐NMR to understand water‐binding food properties

TD‐NMR to understand water‐binding food properties

Adsorption of β-lactoglobulin A on gold surface determined in situ by QCM-D measurements

In this work, we described in detail the physicochemical properties of β-lactoglobulin isoform A (LGB-A) in a bulk solution and on a gold surface. DLS studies confirmed that the LGB trend for aggregation and the formation of larger oligomeric forms are dependent on environmental conditions. The isoelectric point of the system was determined using dynamic surface tension measurements and electrophoretic mobility, which are located at pH 5.45 and 4.8, respectively. The use of these methods has provided valuable information about the conformation stability of the LGB molecule in the electrolyte solution. The properties of self-assembling the LGB-A layer adsorbed on the gold surface were estimated in situ using a quartz crystal microbalance with dissipation monitoring (QCM-D) and Fourier transform infrared spectroscopy (FTIR) as a function of protein concentration, pH, and ionic strength. Based on the measurement of changes in the surface tension at the gas/liquid interface as well as the efficiency of adsorption at the liquid/solid interface using the QCM-D method, we estimated the preferred side-on orientation of the LGB-A molecules. The effectiveness of LGB-A adsorption on the gold surface was mostly controlled by electrostatic interactions that were confirmed by a very strong correlation with the zeta potential of the protein molecule and the gold surface. FTIR spectra shows significant changes in the secondary structure of the adsorbed protein. Particular changes concern the content in the structure β-sheet and random coil forms, with the α-helix content unchanged. The behaviour of the LGB molecule at the interface is in accordance with the direction of the dipole moment of the molecule as determined by molecular dynamics (MD).

Globular bundles and entangled network of proteins (CorA) by a coarse-grained Monte Carlo simulation

Using a coarse-grained model, self-organized assembly of proteins (e.g., CorA and its inner segment iCorA) is studied by examining quantities such as contact profile, radius of gyration, and structure factor as a function of protein concentration at a range of low (native phase) to high (denature phase) temperatures. Visual inspections show distinct structures, i.e., isolated globular bundles to entangled network on multiple length scales in dilute to crowded protein concentrations. In native phase, the radius of gyration of the protein does not vary much with the protein concentration while that of its inner segment increases systematically. In contrast, the radius of gyration of the protein shows enormous growth with the concentration due to entanglement while that of the inner segment remains almost constant in denatured phase. The multi-scale morphology of the collective assembly is quantified by estimating the effective dimension D of protein from scaling of the structure factor: collective assembly from inner segments remains globular (D~3) at almost all length scales in its native phase while that from protein chains shows sparsely distributed morphology with D ≤ 2 in entire temperature range due to entanglement except in crowded environment at low temperature where D~2.6. Higher morphological response of chains with only the inner-segments due to selective interactions in its native phase may be more conducive to self-organizing mechanism than that of the remaining segments of the protein chains.

Assessment of interfacial viscoelastic properties of Faba bean (Vicia faba) protein-adsorbed O/W layers as a function of pH

Interfacial rheology may be regarded as a powerful tool dominating the dynamics of complex fluid-fluid interfaces. More specifically, rheological properties from shear measurements have been postulated as the most useful technique for the assessment of the microstructure of complex fluid-fluid interfaces, and its relationship to long-term emulsion stability. The aim of this work was to evaluate the interfacial properties of Faba bean (FB) protein as a function of protein concentration and pH value (3.0, 5.0 and 8.0). The comparison between results obtained from dilatational and interfacial shear measurements provides accurate details related to the microstructure of the protein at the O/W interface. Viscoelastic moduli of the protein adsorbed at the O/W interface were obtained through dilatational measurements (Tracker, IT Concept), as well as interfacial SAOS measurements, using a double wall-ring geometry fitted to a DHR3 rheometer (TA Intruments). Results from dilatational rheology indicate that proteins are able to form a film at O/W interface, whose strength depends on the pH value (where E’ ranges from 2.0·10−2 Pa m at pH 5.0 to 5.5·10−3 Pa m at pH 8.0). However, these measurements have shown to be less sensitive to pH modifications than interfacial shear viscoelastic measurements. Regardless of the pH value, interfacial shear measurements can follow the evolution of the viscoelastic behaviour of the O/W interfacial layer over the development of the protein gel network, as protein adsorption proceeds. This evolution indicates that protein adsorption takes place much faster at pH 3.0 and 5.0 than at pH 8.0.