Structure Of Β-lactoglobulin Research Articles

Precision fermentation as a route to modify β-lactoglobulin structure through substitution of specific cysteine residues

Precision fermentation is a promising route to develop recombinant proteins with improved functionality. Protein functionality is highly influenced by protein structure, which can be significantly affected by conformational restraints induced by cysteines. Here, we study the impact of individual cysteines on β-lactoglobulin structure, by stepwise substituting them with alanine. Structural characterisation was performed from secondary to quaternary level. The findings demonstrated that the free Cys121 played no role in the folding and dimerisation of BLG. The β-barrel usually includes one disulphide bond (Cys106–Cys119), which was found to be crucial for its formation. The usually exposed disulphide bond (Cys66–Cys160) played no major role in folding of the β-barrel, but mainly modulated its accessibility and mediated dimerisation. Thereby, cysteine mutations can be used as a tool to modify protein structures. These different structures can now be used to further understand the impact on protein behaviour and their functionality.

The attenuating ability of deep eutectic solvents towards the carboxylated multiwalled carbon nanotubes induced denatured β-lactoglobulin structure.

The stabilization of proteins has been a major challenge for their practical utilization in industrial applications. Proteins can easily lose their native conformation in the presence of denaturants, which unfolds the protein structure. Since the introduction of deep eutectic solvents (DESs), there are numerous studies in which DESs act as promising co-solvents that are biocompatible with biomolecules. DESs have emerged as sustainable biocatalytic media and an alternative to conventional organic solvents and ionic liquids (ILs). However, the superiority of DESs over the deleterious influence of denaturants on proteins is often neglected. To address this, we present the counteracting ability of biocompatible DESs, namely, choline chloride-glycerol (DES-1) and choline chloride-urea (DES-2), against the structural changes induced in β-lactoglobulin (Blg) by carboxylated multiwalled carbon nanotubes (CA-MWCNTs). The work is substantiated with various spectroscopic and thermal studies. The spectroscopic results revealed that the fluorescence emission intensity enhances for the protein in DESs. Contrary to this, the emission intensity extremely quenches in the presence of CA-MWCNTs. However, in the mixture of DESs and CA-MWCNTs, there was a slight increase in the fluorescence intensity. Circular dichroism spectral studies reflect the reappearance of the native band that was lost in the presence of CA-MWCNTs, which is a good indicator of the counteraction ability of DESs. Further, thermal fluorescence studies showed that the protein exhibited extremely great thermal stability in both DESs as well as in the mixture of DES-CA-MWCNTs compared to the protein in buffer. This study is also supported by dynamic light scattering and zeta potential measurements; the results reveal that DESs were successfully able to maintain the protein structure. The addition of CA-MWCNTs results in complex formation with the protein, which is indicated by the increased hydrodynamic size of the protein. The presence of DESs in the mixture of CA-MWCNTs and DESs was quite successful in eliminating the negative impact of CA-MWCNTs on protein structural alteration. DES-1 proved to be superior to DES-2 over counteraction against CA-MWCNTs and maintained the native conformation of the protein. Overall, both DESs act as recoiling media for both native and unfolded (denatured by CA-MWCNTs) Blg structures. Both the DESs can be described as potential co-solvents for Blg with increased structural and thermal stability of the protein. To the best of our knowledge, this study for the first time has demonstrated the role of choline-based DESs in the mixture with CA-MWCNTs in the structural transition of Blg. The DESs in the mixture successfully enhance the stability of the protein by reducing the perturbation caused by CA-MWCNTs and then amplifying the advantages of the DESs present in the mixture. Overall, these results might find implications for understanding the role of DES-CA-MWCNT mixtures in protein folding/unfolding and pave a new direction for the development of eco-friendly protein-protective solvents.

Insight into the binding behavior, structure, and thermal stability properties of β-lactoglobulin/Amoxicillin complex in a neutral environment

Antibiotics are extensively used in agriculture and husbandry, thus threatening human health due to their direct exposure to dairy products. Therefore, it is essential to investigate whether milk proteins are unintended carriers of antibiotics. In this research, therefore, we studied the impact of Amoxicillin on the conformation and structural stability of whey protein β-lactoglobulin by using spectroscopic techniques combined with molecular docking and simulation approaches. Further, the thermal stability of the resultant complex was explored. Amoxicillin was observed to effectively quench β-lactoglobulin's intrinsic fluorescence, suggesting that Amoxicillin was successfully bound to the β-lactoglobulin and a stable complex between them was established. The thermodynamic parameters demonstrated that van der Waals forces and hydrogen bonds dominated the spontaneous bonding process. According to circular dichroism (CD) and Fourier transform infrared (FTIR) analyses, the conformational structure of the β-lactoglobulin was altered upon the complexation with Amoxicillin. Further, this interaction significantly enhanced the thermal stability of β-lactoglobulin compared to sole β-lactoglobulin solutions. Molecular docking theoretically showed the preferred binding locus of Amoxicillin within the β-lactoglobulin structure. Molecular dynamics (MD) simulation results further validated the stability of the β-lactoglobulin-Amoxicillin complex. The findings obtained here are helpful for elucidating the potential of β-lactoglobulin to bind and transport harmful substances into the body, thus opening up new avenues for food security.

Adsorption of proteins on TiO2 particles influences their aggregation and cell penetration

TiO2 nanoparticles known as E171 are one controversial food additive due to its potential toxicity. In this work, the main hypothesis is that the proteins adsorbed on the TiO2 nanoparticles prevent their aggregation and favor the cell penetration. To do so, the TiO2 nanoparticles were coated with gelatin and β-lactoglobulin to reach interfacial concentrations about 0.25 mg/mg and 0.32 mg/mg, respectively. The measurement of NP size showed that the protein coating improve the colloidal stability of TiO2 nanoparticles. The FTIR analysis suggests that the β-lactoglobulin structure is modified after adsorption. The penetration of TiO2 penetration inside human intestinal epithelial cells was shown and quantify by using confocal Raman microscopy. The promoting role of the protein coating on the cell penetration was demonstrated for both the gelatin and β-lactoglobulin. Finally, the results allow establishing a correlation between the ability of proteins to prevent NP aggregation and the cell penetration.

Crocin inhibits urea-induced amyloid formation by bovine β-lactoglobulin

Crocin stabilizes the native structure of β-lactoglobulin and attenuates urea-induced unfolding and loss of β-sheet structure during amyloidogenesis.

The impact of ultrasound duration on the structure of β-lactoglobulin

Abstract This study aimed to investigate how ultrasound treatment for different durations (0, 10, 20, 30, 60, and 90 min) impacts the hydrolysis and structural characteristics of β-lactoglobulin (β-LG). Ultrasound conditions were set at ultrasound frequency (40 ± 2) kHz, power 600 W, power density 111.1 W/L, pulse on/off time 10 s/3 s, scanning period 500 ms, and temperature 25 °C. β-LG was pretreated with ultrasound and hydrolyzed by alcalase. Ultrasound treatment for 20 and 30 min significantly improved the degree of hydrolysis and peptide yield; longer treatment durations (60 and 90 min) had little effect on these parameters. Ultrasound treatment caused dynamic changes in protein structure. After a 30-min ultrasound treatment, the particle size, free sulfhydryl groups, intrinsic fluorescence, surface roughness, α-helix, and β-turns increased, whereas absolute zeta potential, disulfide bonds, and β-sheets decreased. When the duration of treatment increased from 30 min to 90 min, the opposite trend was observed. Short-duration ultrasound treatment induced partial unfolding of β-LG, while long-duration treatment induced protein aggregation. Therefore, physicochemical and structural changes in β-LG are largely dependent on ultrasound treatment duration.

Investigation of the effect of oxidation on the structure of β-lactoglobulin by high resolution mass spectrometry

In this work, high-resolution mass spectrometry was used to identify the oxidation sites and forms of β-lactoglobulin (β-Lg) induced by hydrogen peroxide with 1.5% concentration, and the influence of oxidation sites on the structure of β-Lg was discussed from the molecular level. Twelve kinds of oxidation products and 36 oxidation sites were identified, including sulfoxidation in sulfur-containing amino acid residue, hydroxylation in aromatic group residue, deamination in amino-containing amino acid etc. The destruction of hydrogen bonds and disulfide bonds in β-Lg caused by oxidation is the main factor causing its structural changes, which were manifested in the decrease of β-sheet component and increase of β-turns and random coil contents, intrinsic fluorescence intensity and surface hydrophobicity. In addition, several peptides as potential oxidative markers were found to be capable of monitoring the degree of oxidation of β-Lg. In short, this work provided insights into structural changes of β-Lg by oxidation.

Delineating the conformational dynamics of intermediate structures on the unfolding pathway of β-lactoglobulin in aqueous urea and dimethyl sulfoxide

The funnel shaped energy landscape model of the protein folding suggests that progression of folding proceeds through multiple pathways, having the multiple intermediates which leads to multidimensional free-energy surface. Herein, we applied all-atom MD simulation to conduct a comparative study on the structure of β-lactoglobulin (β-LgA) in aqueous mixture of 8 M urea and 8 M dimethyl sulfoxide (DMSO), at different temperatures. The cumulative results of multiple simulations suggest a common unfolding pathway of β-LgA, occurred through the stable and meta-stable intermediates (I), in both urea and DMSO. However, the free-energy landscape (FEL) analyses show that the structural transitions of I-states are energetically different. In urea, FEL shows distinct ensemble of intermediates, I1 and I2, separated by the energy barrier of ∼3.0 kcal mol−1. Similarly, we find the population of two distinct I1 and I2 states in DMSO, however, the I1 appeared transiently around ∼30–35 ns and is short-lived. But, the I2 ensemble is observed structurally compact and long-lived (∼50–150 ns) as compared to unfolding in urea. Furthermore, the I1 and I2 are separated through a high energy barrier of ∼6.0 kcal mol−1. Thus, our results provide the structural insights of intermediates which essentially bear the signature of a different unfolding pathway of β-LgA in urea and DMSO.Abbreviationsβ-LgAβ-lactoglobulinDMSOdimethyl sulfoxideFELfree-energy landscapeGdmClguanidinium chlorideIintermediate stateMGmolten globule statePMEparticle mesh EwaldQfraction of native contactsRMSDroot mean square deviationRMSFroot mean square fluctuationRgradius of gyrationSASAsolvent Accessible Surface AreascSASAthe side chain SASATrptryptophanCommunicated by Ramaswamy H. Sarma

Plasma suPAR in differentiating malignant and benign pancreatic masses.

A number of studies were devoted to understanding an immunological effect of pressure-treated β-lactoglobulin. In our previous work we have proved that high pressure significantly modifies β-lactoglobulin conformation and consequently its physicochemical properties. Here, structure of β-lactoglobulin complex with myristic acid determined at the highest accepted by the crystal pressure value of 550 MPa is reported. Our results structurally prove that pressure noticeably modifies positions of the major β-lactoglobulin epitopes. Considering the biological impact of observed changes in epitope regions, high pressure β-lactoglobulin structure presents a step forward in understanding the pressure modification of food protein allergenicity. The conformational changes of pressurized β-lactoglobulin did not support the hypothesis that proteolytic digestion facilitated by pressure is caused by an exposure of the digestive sites. Our findings demonstrate that high pressure protein crystallography can potentially identify the most pressure-sensitive fragments in allergens, and can therefore support development of hypoallergenic food products.

Effect of Methylglyoxal-Induced Glycation on the Composition and Structure of β-Lactoglobulin and α-Lactalbumin.

Glycation, and particularly reactions between aldehydes and nucleophiles (thiols, amines), can initiate changes in the structure, solubility, composition, hydrophobicity, conformation, function, and susceptibility to proteolysis of proteins. This can have adverse consequences for mammals, plants, foodstuffs, and pharmaceuticals. Low-molecular-mass dialdehydes such as methylglyoxal (MGO) are much more reactive than parent glucose and therefore potentially highly damaging. These are present at significant levels in some foods. This study investigated whether and how MGO exposure, with or without concurrent heat exposure, affected the major whey proteins β-lactoglobulin and α-lactalbumin. MGO diminished the formation of heat-induced, reducible, intermolecular disulfide cross-links for both proteins, with this being associated, at least in part, with alternative thiol consuming reactions of MGO. At long incubation times, nonreducible protein cross-links were formed in a dose-dependent manner, with LC-MS/MS and UPLC analysis showing the presence of methylglyoxal-lysine dimers (MOLD). UPLC analysis revealed MGO-dependent consumption of specific amino acids in the order Cys > Arg > Lys > Trp for both proteins, with α-lactalbumin affected to a greater extent than β-lactoglobulin. SDS-PAGE revealed altered protein mobility consistent with modification of charged residues. MGO exposure also resulted in increased binding of the hydrophobic dye, 8-anilino-1-naphthalene sulfonic acid, consistent with limited protein unfolding. Overall, these data are consistent with rapid reaction of MGO residues at Cys residues (when available) and surface accessible Arg and Lys residues, with formation of adducts and cross-linked materials. These alternative reactions of dialdehydes diminish direct heat-induced (disulfide) cross-link formation and result in limited protein unfolding.

Towards understanding the effect of high pressure on food protein allergenicity: β-lactoglobulin structural studies

A number of studies were devoted to understanding an immunological effect of pressure-treated β-lactoglobulin. In our previous work we have proved that high pressure significantly modifies β-lactoglobulin conformation and consequently its physicochemical properties. Here, structure of β-lactoglobulin complex with myristic acid determined at the highest accepted by the crystal pressure value of 550 MPa is reported. Our results structurally prove that pressure noticeably modifies positions of the major β-lactoglobulin epitopes. Considering the biological impact of observed changes in epitope regions, high pressure β-lactoglobulin structure presents a step forward in understanding the pressure modification of food protein allergenicity. The conformational changes of pressurized β-lactoglobulin did not support the hypothesis that proteolytic digestion facilitated by pressure is caused by an exposure of the digestive sites. Our findings demonstrate that high pressure protein crystallography can potentially identify the most pressure-sensitive fragments in allergens, and can therefore support development of hypoallergenic food products.

Revealing the Dimeric Crystal and Solution Structure of β-Lactoglobulin at pH 4 and Its pH and Salt Dependent Monomer-Dimer Equilibrium.

The dimeric structure of bovine β-lactoglobulin A (BLGA) at pH 4.0 was solved to 2.0 Å resolution. Fitting the BLGA pH 4.0 structure to SAXS data at low ionic strength (goodness of fit R-factor = 3.6%) verified the dimeric state in solution. Analysis of the monomer-dimer equilibrium at varying pH and ionic strength by SAXS and scattering modeling showed that BLGA is dimeric at pH 3.0 and 4.0, shifting toward a monomer at pH 2.2, 2.6, and 7.0 yielding monomer/dimer ratios of 80/20%, 50/50%, and 25/75%, respectively. BLGA remained a dimer at pH 3.0 and 4.0 in 50-150 mM NaCl, whereas the electrostatic shielding raised the dimer content at pH 2.2, 2.6, and 7.0, i.e., below and above the pI. Overall, the findings provide new insights into the molecular characteristics of BLGA relevant for dairy product formulations and for various biotechnological and pharmaceutical applications.

Changes in structure and antioxidant activity of β-lactoglobulin by ultrasound and enzymatic treatment

Effects of ultrasound (20–40% amplitudes at 45–55 °C) and enzymatic (pepsin and trypsin) treatment on structure and antioxidant activity of β-lactoglobulin were studied. Changes in structure of β-lactoglobulin were investigated using spectroscopy techniques and changes in antioxidant activity were measured by chemical and cellular-based assays. Ultrasound treatment had considerable impact on the structure of β-lactoglobulin and increased the susceptibility of β-lactoglobulin to both pepsin and trypsin proteolysis. Intrinsic fluorescence intensity of β-lactoglobulin was increased by ultrasound and then decreased after following enzymatic treatment. Compared with control, the β-lactoglobulin after ultrasound and enzymatic treatments showed significantly higher oxygen scavenging activities in Caco-2 cells models, ABTS (2, 2′-Azinobis-3-ethylbenzthiazoline-6-sulphonate) radical scavenging activity and oxygen radical absorbance capacity (p < 0.05). Results indicated that ultrasound treatment increased the proteolysis of β-lactoglobulin by both pepsin and trypsin and improved the antioxidant activity of the protein and its proteolytic products.

Effect of ultrasound treatment on antioxidant activity and structure of β-Lactoglobulin using the Box–Behnken design

ABSTRACTEffect of ultrasound treatment on antioxidant activity and structure of β-Lactoglobulin were investigated using antioxidant indexes and spectroscopy techniques. The 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities had been applied to the experimental response obtained by a three-factor-three-level Box–Behnken design. Results showed that ultrasound treatment significantly increased antioxidant activity (p < 0.05). The maximum DPPH radical scavenging activity of β-Lactoglobulin was 57.59% at the ultrasound conditions of 45°C, 20 min and amplitude of 30%. The ultrasound-treated β-Lactoglobulin exhibited higher 2,2ʹ-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical scavenging activity and oxygen radical absorbance capacity. Circular dichroism data indicated that ultrasound treatment increased both α-helix and β-sheet contents of β-Lactoglobulin and altered Trp residues, which resulted in the secondary and tertiary structure changes. Differential scanning calorimeter and scanning electron microscopy micrographs showed that ultrasound-treated β-Lactoglobulin contained larger aggregates than untreated. In conclusion, ultrasound treatment had considerable effects on antioxidant activity and structure of β-Lactoglobulin. Ultrasound treatment may expand the applications of β-Lactoglobulin in food industries.

Effect of high pressure homogenization on the structure and the interfacial and emulsifying properties of β-lactoglobulin

The effect of high pressure homogenization (HPH) on the structure of β-lactoglobulin (β-lg) was studied by combining spectroscopic, chromatographic, and electrophoretic methods. The consequences of the resulting structure modifications on oil/water (O/W) interfacial properties were also assessed. Moderated HPH treatment (100 MPa/4 cycles) showed no significant modification of protein structure and interfacial properties. However, a harsher HPH treatment (300 MPa/5 cycles) induced structural transformation, mainly from β-sheets to random coils, wide loss in lipocalin core, and protein aggregation via intermolecular disulfide bridges. HPH-modified β-lg displayed higher surface hydrophobicity leading to a faster adsorption rate at the interface and an earlier formation of an elastic interfacial film at Cβ-lg = 0.1 wt%. However, no modification of the interfacial properties was observed at Cβ-lg = 1 wt%. At this protein concentration, the prior denaturation of β-lg by HPH did not modify the droplet size of nanoemulsions prepared with these β-lg solutions as the aqueous phases. A slightly increased creaming rate was however observed. The effects of HPH and heat denaturations appeared qualitatively similar, but with differences in their extent.

Monomerization and aggregation of β-lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation

β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~55Å, ~29Å and ~37Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57kcalmol−1 and 8.93kcalmol−1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~77Å and ~117Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69kcalmol−1 and 9.09kcalmol−1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.

Monolayer polymerization of polyhedral oligomeric silsesquioxane on graphene oxide for highly efficient adsorption of β-lactoglobulin

We report the self-assembly of monolayer polyhedral oligomeric silsesquioxane (POSS) on graphene oxide (GO), where GO acts as a directing template along with the assembly of POSS into a three-dimensional porous framework structure, shortly termed as PPG. The PPG framework contains ultrathin nanopetals with a thickness of ca. 1.95 nm, giving rise to reduced restacking and high surface area. The PPG framework exhibits a high adsorption capacity of up to 1570.3 mg g−1 towards β-lactoglobulin, in addition to a favorable selectivity against other protein species with similar isoelectric points to that of β-lactoglobulin. This might be attributed to the specific hydrogen-bonding donor-acceptor interaction between the calyx structure of β-lactoglobulin and the PPG framework. Satisfactory separation performance of PPG is confirmed by the selective discrimination and isolation of β-lactoglobulin from complex biological sample matrixes, e.g., milk whey. This observation provides a promising approach for the construction of monolayer polymer-modified three-dimensional graphene oxide composites with specific biological application potentials.

Complexation of bovine β-lactoglobulin with malvidin-3-O-glucoside and its effect on the stability of grape skin anthocyanin extracts

The binding interaction between bovine β-lactoglobulin and malvidin-3-O-glucoside (MG), the major anthocyanin in grape skin anthocyanin extracts (GSAE), was studied at pH 6.3 using fluorescence, Fourier transform infrared and circular dichroism spectroscopy. The binding constant (KS), binding force and effect of the interaction on the β-lactoglobulin conformation and GSAE stability were investigated. The results indicated that β-lactoglobulin complexed with MG mainly via hydrophobic interaction with KS of 0.67×103M−1 at 297K. The secondary structure of β-lactoglobulin was changed by MG binding, with a decrease in α-helix, turn and random coil and an increase in β-sheet. Bovine whey protein effectively prevented the color fading and degradation of anthocyanin in the GSAE solution during the thermal treatment (80°C/2h), H2O2 oxidation (0.005% H2O2/1h) and photo illumination (5000lx/5d). The whey protein-anthocyanin complexation appeared to have a positive effect on the thermal, oxidation and photo stability of GSAE.

Adsorption behavior of β-lactoglobulin onto polyethersulfone membrane surface

The adsorption of whey protein onto polyethersulfone (PES) membrane was investigated by static adsorption experiments to understand fouling mechanism and optimize the process condition to minimize the membrane fouling. Adsorption isotherm was applied to calculate the isotherm parameters such as adsorption capacity (KF) and surface heterogeneity (1/n). The KF values increased about 3.7 times at pH 3.0, 1.5 times at pH 5.2, and 2.3 times at pH 9.0 compared to that at pH 7.0. The hydrophobic interaction by dissociation of dimer structure of β-lactoglobulin to monomer structure at acidic and alkaline conditions showed the greatly increasing of the amount of protein adsorption by the protein and membrane interaction which might form the strong and rigid protein layer on the polymeric membrane surface. The addition of salt reduced the protein adsorption on PES membrane because of the interactions between charged protein molecules and salt ions.

Effect of Thermal Treatment on Chemical Structure of Β-Lactoglobulin and Basil Seed Gum Mixture at Different States by ATR-FTIR Spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy was used to compare the structure of β-lactoglobulin, basil seed gum, and β-lactoglobulin-basil seed gum mixtures, at different states (powder, solution, and gel). The effects of heating and different ratios of β-lactoglobulin-basil seed gum were also investigated to determine their impact on chemical structure and understand their interaction. The results showed that gelification process proved a pronounced effect upon β-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding β-sheet structure. These results confirmed that this structure may be necessary for the formation of a gel network. Basil seed gum had a distinct peak at around 1603 cm–1 that relates to –COO–1 stretching of carboxylate salts, probably uronic acids, which approved its anionic structure. The Fourier transform infrared spectroscopy findings strongly suggested that these two polymers are thermodynamic incompatible as amide I peak was increased in the β-lactoglobulin-basil seed gum mixed system and carbon–nitrogen (CN) stretching peak was observed at 2125 cm–1. On the basis of these findings, it was possible to modify the ability of β-lactoglobulin-basil seed gum to form a gel and as a consequence to control the gelling and emulsifying properties.