A novel proteomic approach for the identification and relative quantification of disulfide-bridges in the human milk proteome.

This study explores the disulfide bridges present in the human milk proteome by a novel approach permitting both positional identification and relative quantification of the disulfide bridges. Human milk from six donors was subjected to trypsin digestion without reduction. The digested human milk proteins were analyzed by nanoLC-timsTOF Pro combined with data analysis using xiSEARCH. A total of 85 unique disulfide bridges were identified in 25 different human milk proteins. The total relative abundance of disulfide bridge-containing peptides constituted approximately 5% of the total amount of tryptic-peptides. Seven inter-molecular disulfide bridges were identified between either α-lactalbumin and lactotransferrin (5) or αS1-casein and κ-casein (2). All cysteines involved in the observed disulfide bridges of α-lactalbumin and lactotransferrin were mapped onto protein models using AlphaFold2 Multimer to estimate the length of the observed disulfide bridges. The lengths of the disulfide bridges of lactotransferrin indicate a potential for multi- or poly-merization of lactotransferrin. The high number of intramolecular lactotransferrin disulfide bridges identified, suggests that these are more heterogeneous than previously presumed. SIGNIFICANCE: Disulfide-bridges in the human milk proteome are an often overseen post-transaltional modification. Thus, mapping the disulfide-bridges, their positions and relative abundance, are valuable new knowledge needed for an improved understanding of human milk protein behaviour. Although glycosylation and phosphorylation have been described, even less information is available on the disulfide bridges and the disulfide-bridge derived protein complexes. This is important for future work in precision fermentation for recombinant production of human milk proteins, as this will highlight which disulfide-bridges are naturally occouring in human milk proteins. Further, this knowledge would be of value for the infant formula industry as it provides more information on how to humanize bovine-milk based infant formula. The novel method developed here can be broadly applied in other biological systems as the disulfid-brigdes are important for the structure and functionality of proteins.

Myricetin and morin hydrate inhibit amyloid fibril formation of bovine α-lactalbumin (BLA).

Amyloid fibrils are self-assembled aggregates of proteins and peptides that can lead to a broad range of diseases called amyloidosis. So far, no definitive and approved treatment to target directly amyloid fibrils has been introduced. Nevertheless, the search for small molecules with ability to inhibit and suppress fibril formation is an active and promising area of the research. Herein, the binding interactions and inhibitory effects of myricetin and morin hydrate on the in vitro fibrillation of bovine α-lactalbumin (BLA) have been investigated. The intrinsic fluorescence of BLA was quenched by myricetin and morin hydrate through combination of the static and dynamic quenching along with non-radiative Förster energy transfer mechanisms. The binding of these two flavonoids to BLA were not accompanied by major alteration in the conformation of BLA as evidenced by CD studies. The results of the fluorescence quenching analyses indicated almost the same binding affinities of myricetin and morin hydrate toward BLA (Kb ~ 106 M-1). However, the results of thioflavin T (ThT) assays showed that myricetin is a stronger inhibitor against BLA fibrillation compared to morin hydrate.

HAMLET in human milk is resistant to digestion and carries essential free long chain polyunsaturated fatty acids and oleic acid.

The oleic acid/alpha-lactalbumin complex HAMLET (human alpha-lactalbumin made lethal to tumors) is cytotoxic to various cancerous cell lines and is assembled from alpha-lactalbumin (ALA) and free oleic acid (OA). HAMLET is also cytotoxic to normal immature intestinal cells. It remains unclear if HAMLET, experimentally assembled with OA and heat, can spontaneously assemble in frozen human milk over time. To approach this issue, we used a set of timed proteolytic experiments to evaluate the digestibility of HAMLET and native ALA. The purity of HAMLET in human milk was confirmed by ultra high performance liquid chromatography coupled to tandem mass spectrometry and western blot to resolve the ALA and OA components. Timed proteolytic experiments were used to identify HAMLET in whole milk samples. Structural characterization of HAMLET was performed by Fournier transformed infrared spectroscopy and indicated a transformation of secondary structure with increased alpha-helical character of ALA upon binding to OA.

Purification and identification of xanthine oxidase inhibitory peptides from enzymatic hydrolysate of α-lactalbumin and bovine colostrum casein.

The development of food-derived Xanthine Oxidase (XO) inhibitors is critical to the treatment of hyperuricemia and oxidative stress-related disease. Few studies report on milk protein hydrolysates' XO inhibitory activity, with the mechanism of their interaction remaining elusive. Here, different commercial enzymes were used to hydrolyze α-lactalbumin and bovine colostrum casein. The two proteins hydrolyzed by alkaline protease exhibited the most potent XO inhibitory activity (bovine casein: IC50 = 0.13 mg mL-1; α-lactalbumin: IC50 = 0.28 mg mL-1). Eight potential XO inhibitory peptides including VYPFPGPI, GPVRGPFPIIV, VYPFPGPIPN, VYPFPGPIHN, QLKRFSFRSFIWR, LVYPFPGPIHN, AVFPSIVGR, and GFININSLR (IC50 of 4.67-8.02 mM) were purified and identified from alkaline protease hydrolysates by using gel filtration, LC-MS/MS and PeptideRanker. The most important role of inhibiting activity of peptides is linked to hydrophobic interactions and hydrogen bonding based on the results of molecular docking and molecular dynamics simulation. The enzymatic hydrolysate of α-lactalbumin and bovine colostrum casein could be a competitive candidates for hyperuricemia-resisting functional food.

Physical treatment synergized with natural surfactant for improving gas-water interfacial behavior and foam characteristics of α-lactalbumin.

The purpose of this study was to investigate effect of physical treatment (ultrasound, U/high pressure homogenization, H/combined treatment, UH or HU) and surfactant (Mogroside V, Mog) on air/water interface adsorption and foaming properties of α-lactalbumin (ALa). Firstly, the binding of Mog and all physical-treated ALa was a static quenching process. Mog had the greatest binding affinity for HU-ALa among all treated samples. U or H treatment could change surface hydrophobicity of ALa/Mog complex. Secondly, at the molar ratio (ALa:Mog) of 1:50, foaming ability (FA) of all ALa samples got the maximum. The sequence of FA in ALa and ALa/Mog complex was listed as follow: HU > U > H > UH. Moreover, foaming stability (FS) of HU-ALa was the highest, followed by H-ALa, U-ALa and UH-ALa. Meanwhile, low concentration Mog increased FS of ALa or UH-ALa, but it reduced FS of H-ALa, U-ALa and HU-ALa. Quartz crystal microbalance with dissipation monitoring (QCM-D) experiment indicated that ALa/Mog complex after U or H treatment was quickly absorbed at air/water interface, compared with the treated ALa, and HU-ALa/Mog had the largest frequency shift. In addition, HU-ALa had the thickest bubble membrane and the highest dissipation shift in all samples, indicating that the absorbed membrane thickness and viscoelasticity of samples was correlated with foam stability. Therefore, U and H treatment synergism with Mog was an effective approach to enhance foam properties of ALa, which indicated that HU-treated ALa/Mog complex could be viewed as the safe and efficient foaming agent applied in food processing.

Calcium bioaccessibility increased during gastrointestinal digestion of α-lactalbumin and ß-lactoglobulin.

Calcium bioaccessibility depends on the amount of soluble calcium under intestinal digestion. The changes in calcium during in vitro static digestion of α-lactalbumin and ß-lactoglobulin in presence of calcium chloride (0 mM, 20 mM and 50 mM) were followed by combining electrochemical determination of free calcium with the determination of soluble calcium by inductively coupled plasma optical emission spectroscopy. α-Lactalbumin and, more evident, ß-lactoglobulin were found to increase calcium bioaccessibility with increasing intestinal digestion time by around 5% and 10%, respectively, due to the complex binding of calcium to peptides formed from protein hydrolysis by gastrointestinal enzymes. In vitro digested samples of ß-lactoglobulin in presence of CaCl2 had nearly twice as much complex bound calcium as α-lactalbumin samples. The calcium bioaccessibility decreased significantly with the increasing concentration of added calcium chloride, although the amount of calcium chloride had little effect on the extension of digestion of α-lactalbumin and ß-lactoglobulin. Simulated digestion fluids were found to have a negative effect on calcium bioaccessibility, especially the presence of hydrogen phosphate, and the amount of precipitated calcium increased significantly with increasing amount of added calcium chloride. Based on analysis and visualization by sequences of the peptides formed during digestion of α-lactalbumin and ß-lactoglobulin, it was observed that peptides containing aspartic acid and glutamic acid acting as calcium chelators, may prevent precipitation of calcium in the intestines and increase calcium bioaccessibility. These results provide knowledge for the design of new dairy based functional foods to prevent calcium deficiency.

Ultrasound-assisted glycation and the allergenicity of α-lactalbumin.

BACKGROUND: Ultrasound-assisted glycation is a promising method for decreasing the allergenicity of α-lactalbumin (ALA). However, there is a lack of in vivo studies on the allergenicity of ultrasound-assisted glycated ALA. In this study, the effects of the ultrasound-assisted glycation of ALA on the allergenicity and intestinal microflora were characterized using a BALB/c mouse model. RESULTS: Increased immunoglobulin -G/ immunoglobulin-E (IgG/IgE) and interleukin-4/6 (IL-4/6) secretions, and reduced interferon-γ (IFN-γ) secretions were found in the serum of ALA sensitized and challenged, mice in comparison with a control group. However, there was no significant difference between the mice fed with ultrasound-assisted glycated ALA and the control group. Mice that were sensitized and challenged with ALA showed disrupted intestinal microflora, manifesting in significantly decreased Firmicutes and significantly increased Proteobacteria. It was found that 100ALA-gal could maintain the intestinal microflora of mice in a normal state. Pearson's rank correlation showed that Proteobacteria and Spirochaetota were correlated positively with the IL-4/IL-6 level and were correlated negatively with the expression of IFN-γ. Proteobacteria were also significantly positively correlated with IL-6 and negatively correlated with IFN-γ (P < 0.05). CONCLUSION: These results suggested that ultrasound-assisted glycation on ALA can maintain the intestinal microflora in a normal state thus balancing the proportion of Th1/Th2 to decrease allergic reaction. © 2022 Society of Chemical Industry.

Comparison of interaction, structure, and cell proliferation of α-lactalbumin-safflower yellow complex induced by microwave heating or conventional heating.

BACKGROUND: The protein-polyphenol interaction mechanism has always been a research hotspot, but their interaction is affected by heat treatment, which is widely applied in food processing. Moreover, the effects of microwave or water-bath heating on the protein-polyphenol interaction mechanism have been not clarified. The pasteurization condition (65 °C, 30 min) was selected to compare the effects of microwave or water bath on binding behavior, structure, and cell proliferation between α-lactalbumin (α-LA) and safflower yellow (SY), thus providing a guide for the selection of functional dairy processing conditions. RESULTS: Microwave heat treatment of α-LA-SY resulted in stronger fluorescence quenching than that of conventional heat treatment. Moreover, the binding constant Ka of all α-LA-SY samples was augmented significantly after microwave or water bath treatment, and microwave-heated α-LA-SY showed the maximum Ka . Fourier transform infrared spectroscopy showed that microwave heating resulted in more ordered structures of α-LA into its disordered structures than water bath heating. However, the ferric reducing antioxidant power and chroma value of α-LA-SY were more reduced by microwave heating than by water bath heating. Moreover, microwave heating facilitated the cell proliferation of α-LA-SY compared with water bath treatment. CONCLUSION: It was demonstrated that microwave heating promoted interaction between α-LA and SY more than water bath heating did. Microwave heat treatment was a safe and effective way to enhance the binding affinity of α-LA to SY, being a potential application in food industry. © 2022 Society of Chemical Industry.

The influence of cations on α-lactalbumin amyloid aggregation.

There is limited knowledge regarding α-lactalbumin amyloid aggregation and its mechanism. We examined the formation of α-lactalbumin amyloid fibrils (α-LAF) in the presence of cations (Mg²⁺, Ca²⁺, Na⁺, K⁺, NH₄⁺, and Cs⁺) in the form of chloride salts at two concentrations. We have shown that studied cations affect the conformation of α-lactalbumin, the kinetics of its amyloid formation, morphology, and secondary structure of α-LAF in a different manner. The higher salts concentration significantly accelerated the aggregation process. Both salt concentrations stabilized α-lactalbumin's secondary structure. However, the presence of divalent cations resulted in shorter fibrils with less ß-sheet content. Moreover, strongly hydrated Mg²⁺ significantly altered α-lactalbumin's tertiary structure, followed by Na⁺, NH₄⁺, K⁺, and weakly hydrated Cs⁺. On the other hand, Ca²⁺, despite being also strongly hydrated, stabilized the tertiary structure, supposedly due to its high affinity towards α-lactalbumin. Yet, Ca²⁺ was not able to inhibit α-lactalbumin amyloid aggregation.

The kinetic mechanism of cations induced protein nanotubes self-assembly and their application as delivery system.

The amphiphilic proteins can be used as building blocks (BBs) forming various self-assemblies. Understanding their self-assembly mechanism is important for designing novel nanomaterials. Herein, the BBs dimers were first prepared from carboxyl-abundant enzymolyzed α-lactalbumin (α-lac) at 50 °C. Then the unidentate coordination of Ca2+ between the BBs caused a ß-sheet stacking to further self-assemble into nanotubes (NTs). Compared with the traditional "one-pot" method, a step-wise new method was applied to study hydrolysis, aggregation and self-assembly processes separately. The α-lac was hydrolyzed into 11 kDa amphiphilic peptides independent of temperature while a BBs dimer was formed at 50 °C by hydrophobic interaction. Ca2+ induced a conformational change of BBs and promoted these BBs gradually aggregate into 10 strands of filaments, which twisted into helical ribbons by electrostatic repulsion. Ca2+ further induced the twisted helical ribbons closed into NTs driven by the reduction of line tension energy. Besides, the carboxyl-Ca2+ coordination dominated NTs elongation in the longitudinal direction and filaments aggregation in the lateral direction with the same binding stoichiometry of 1:1 respectively. Finally, NTs successfully encapsulated curcumin and improved the viscosity of liquid food. α-Lac NTs show a high potential as a delivery system for food applications.

Isolation and allergenicity evaluation of glycated α-lactalbumin digestive products and identification of allergenic peptides.

This study aimed to isolate and evaluate the allergenicity of glycated α-lactalbumin (ALA) digestive products and identify its allergenic peptides. The digestive products of native-, alone glycated- and ultrasound-assisted glycated ALA (ALA-D, ALA-gal-D, 100ALA-gal-D) were isolated into three fractions (F1, F2 and F3). High-resolution mass spectrometry showed that the digestion-resistant peptides of F2 and F3 mainly distributed in amino acid sequence (AA) 25-31, AA32-53, AA40-53, AA54-60, AA80-90, AA94-104. The allergenicity of the three fractions of glycated ALA was lower than that in ALA-D, indicating glycation of ALA could indeed reduce its allergenicity after digestion. Furthermore, most fractions isolated from high glycation-degree ALA had the lowest allergenicity. The IgG/IgE binding abilities of synthesized peptides indicated that AA94-104 firstly identified by us embodied the strongest allergenicity and might be the potential allergenic peptide. This will provide a theory for preparing hypoallergenic products based on the identified allergenic peptides.

Lipid bilayer composition as a determinant of cancer cell sensitivity to tumoricidal protein-lipid complexes.

Complexes formed by the alpha1 N-terminal peptide of alpha-lactalbumin and oleic acid (alpha1-oleate) interact with lipid bilayers. Plasma membrane perturbations trigger tumor cell death but normal differentiated cells are more resistant, and their plasma membranes are less strongly affected. This study examined membrane lipid composition as a determinant of tumor cell reactivity. Bladder cancer tissue showed a higher abundance of unsaturated lipids enriched in phosphatidylcholine, PC (36:4) and PC (38:4), and sphingomyelin, SM (36:1) than healthy bladder tissue, where saturated lipids predominated and the lipid extracts from bladder cancer tissue inhibited the tumoricidal effect of the complex more effectively than healthy tissue extracts. Furthermore, unsaturated PC in solution inhibited tumor cell death, and the complex interacted with giant unilamellar vesicles formed by PC, confirming the affinity of alpha1-oleate for fluid membranes enriched in PC. Quartz Crystal Microbalance with dissipation monitoring (QCM-D) detected a preference of the complex for the liquid-disordered phase, suggesting that the insertion into PC-based membranes and the resulting membrane perturbations are influenced by membrane lipid saturation. The results suggest that the membrane lipid composition is functionally important and that specific unsaturated membrane lipids may serve as "recognition motifs" for broad-spectrum tumoricidal molecules such as alpha1-oleate.

Oxidation of Whey Proteins during Thermal Treatment Characterized by a Site-Specific LC-MS/MS-Based Proteomic Approach.

Thermal treatment is often employed in food processing to tailor product properties by manipulating the ingredient functionality, but these elevated temperatures may accelerate oxidation and nutrient loss. Here, oxidation of different whey protein systems [α-lactalbumin (α-LA), ß-lactoglobulin (ß-LG), a mix of α-LA and ß-LG (whey model), and a commercial whey protein isolate (WPI)] was investigated during heat treatment at 60-90 °C and a UHT-like treatment by LC-MS-based proteomic analysis. The relative modification levels of each oxidation site were calculated and compared among different heat treatments and sample systems. Oxidation increased significantly in protein systems after heating at ≥90 °C but decreased in systems with higher complexity [pure protein (α-LA > ß-LG) > whey model > WPI]. In α-LA, Cys, Met, and Trp residues were found to be most prone to oxidation. In ß-LG-containing protein systems, Cys residues were suggested to scavenge most of the reactive oxidants and undergo an oxidation-mediated disulfide rearrangement. The rearranged disulfide bonds contributed to protein aggregation, which was suggested to provide physical protection against oxidation. Overall, limited loss of amino acid residues was detected after acidic hydrolysis followed by UHPLC analysis, which showed only a minor effect of heat treatment on protein oxidation in these protein systems.

An able-cryoprotectant and a moderate denaturant: distinctive character of ethylene glycol on protein stability.

Osmolytes are known to stabilize proteins against denaturing conditions. Ethylene glycol (EG), however, shows a distinctive effect on α-lactalbumin (α-LA) that it stabilizes the protein against cold-induced denaturation, whereas it destabilizes during heat denaturation. The replica exchange molecular dynamics (REMD) simulation of α-LA in the presence of EG shows that EG denatures the protein at higher temperatures whereas it retards the denaturation at sub-zero temperature. Representative structures of α-LA were selected from REMD trajectories at three different temperature conditions (240, 300 and 340 K) with and without EG, and classical molecular dynamics (MD) simulations were performed. The results suggest that the presence of water around α-LA is more at lower temperatures; however, water around the hydrophobic residues is reduced with the addition of EG at sub-zero temperature. The partition coefficient of EG showed that the binding of EG with hydrophobic residues was higher at lower temperatures. Preferential interaction parameters at different temperatures were calculated based on the mean distribution (Γ23) and Kirkwood-Buff integral (G23) methods. Γ23 shows a larger positive value at 240 K compared to higher temperatures. G23 shows positive values at lower temperatures, whereas it becomes negative at above 280 K. These results indicate that the preferential binding of EG with α-LA is more at sub-zero temperature compared to higher temperature conditions. Thus, the study suggests that the preferential binding of EG reduces the hydrophobic hydration of α-LA at lower temperatures, and stabilizes the protein against cold denaturation. However, the preferential binding of EG at higher temperature drives the folding equilibrium towards the denatured state.Communicated by Ramaswamy H. Sarma.

UHT treatment and storage of liquid infant formula affects protein digestion and release of bioactive peptides.

Ready-to-feed liquid infant formulas (IF) were subjected to direct (D) or indirect (ID) ultra-high-temperature (UHT) treatment and then stored at 40 °C under aseptic conditions for 60-120 days simulating global transportation which accelerates the Maillard reaction. Low pasteurized and unstored IF (LP) was included as a control for the UHT treatments. Simulated infant in vitro digestion was conducted. SDS-PAGE indicated that protein aggregate formation correlated with thermal treatment, being greatest after 60 days of storage. Limited protein digestion was observed after pepsin treatment for 2 h. Beta-lactoglobulin (ß-Lg), alpha-lactalbumin (α-La) and protein aggregates remained undigested after 2 h of pepsin digestion in LP and D, but less ß-Lg and α-La remained in ID. The digestion of ß-Lg and α-La was enhanced in D and ID stored for 60 days, but aggregates remained undigested. After pepsin and pancreatin digestion, large amounts of ß-Lg remained undigested in the LP, but digestion increased after UHT treatment (ID > D) and increased further after storage for 60 and 120 days, indicating that heat treatment and storage facilitate the digestion of unaggregated proteins. No aggregates remained after pancreatin digestion of LP, D, ID and D stored for 60 days, but were present in ID stored for 60 days. Aggregates were mainly disulphide-linked, but dityrosine linkages were detected in D and ID stored for 120 days. LC-MS/MS indicated limited proteolysis arising from endogenous milk proteases prior to in vitro digestion, being highest in D. Peptide numbers increased following pepsin and further during pancreatin digestion (ß-casein > ß-Lg > ß-La), and released ß-Lg peptides, typically 5-8 amino acids in length, contained several bioactivities, e.g., dipeptidyl-peptidase IV (DPP-IV) and angiotensin converting enzyme (ACE) inhibition.

Protection of menhaden oil from oxidation in Pickering emulsion-based delivery systems with α-lactalbumin-chitosan colloidal nanoparticle.

In this study, α-lactalbumin-chitosan (ALA-CHI) colloidal nanoparticles were spontaneously formed mainly through electrostatic interactions for stabilizing Pickering emulsion loaded with health-beneficial but unstable menhaden oil. The oxidative stability of menhaden oil was supposed to be significantly enhanced with Pickering emulsion-based delivery systems with ALA-CHI colloidal particles. The film of ALA-CHI colloidal nanoparticles had higher surface hydrophobicity than ALA at pH 5.0, and 6.5. A near-neutral wettability (89.6°) of ALA-CHI nanoparticles was observed at pH 5.0. Stable Pickering emulsions (60% menhaden oil fraction, w/w) were successfully fabricated with only 0.12% (w/w) of ALA-CHI nanoparticles. Pickering emulsions exhibited superior storage, heat, and centrifugation stability. The formation of gel-like structures was confirmed by rheological results. The viscosity and storage modulus in the frequency range of 0.1 to 10 Hz with a 1.0% strain exhibited remarkable increases with increasing colloidal particle concentration or oil fraction. Increasing oil fractions from 20% to 60% (w/w) or colloidal particle concentrations 0.12% to 2.40% (w/w) can pronouncedly facilitate the inhibition of lipid oxidation, as confirmed by detecting the formation of primary and secondary oxidation products.

Identification and characterization of antioxidant peptides obtained from the bioaccessible fraction of α-lactalbumin hydrolysate.

Whey is an abundantand sustainable source of bioactive peptides obtained from cheese making process. Whey proteins such as α-lactalbumin can be biologically active when the bioactive peptides encrypted in the amino acid sequence of the native protein are released by enzymatic hydrolysis. In the present work, the identification, sequence analysis, and antioxidant activity of bioaccessible peptides from α-lactalbumin alcalase-hydrolysate was assessed. Antioxidant activity (ABTS, ORAC, and HORAC) of α-lactalbumin showed a significant increase (p < 0.05) after the enzymatic treatment with alcalase and this capacity increased even more after the simulation of the gastrointestinal digestion process. Peptides contained in the gastrointestinal digest of α-lactalbumin hydrolysate were separated by preparative RP-HPLC (55 fractions), and three peptides were identified by LC-MS/MS analysis from selected fractions: IWCKDDQNPH (MW: 1254.54 Da) f(59-68), KFLDDDLTDDIM (MW: 1439.64 Da) f(79-90), DKFLDDDLTDDIM (MW: 1554.67 Da) f(78-90). Among the chemically synthesized peptides, IWCKDDQNPH showed the highest antioxidant capacity determined by ORAC, ABTS, and HORAC assays (IC50 0.015 ± 0.002, 0.45 ± 0.02, and 1.30 ± 0.05 mg/ml, respectively) and this activity may be related to the amino acid sequence. This is the first report where these bioaccessible peptides from α-lactalbumin hydrolysate were identified. The α-lactalbumin hydrolysate could be employed as a functional antioxidant ingredient. PRACTICAL APPLICATION: The present work studied the bioaccessibility of antioxidant peptides from an α-lactalbumin alcalase-hydrolysate by identifying three novel bioaccessible peptides responsible for the antioxidant capacity, providing evidence of the hydrolysate potential as an antioxidant ingredient in the formulations of functional foods and/or food supplements.

Investigating the Disordered and Membrane-Active Peptide A-Cage-C Using Conformational Ensembles.

The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers.

Milk Proteins-Their Biological Activities and Use in Cosmetics and Dermatology.

Milk and colostrum have high biological potential, and due to their natural origin and non-toxicity, they have many uses in cosmetics and dermatology. Research is ongoing on their potential application in other fields of medicine, but there are still few results; most of the published ones are included in this review. These natural products are especially rich in proteins, such as casein, ß-lactoglobulin, α-lactalbumin, lactoferrin, immunoglobulins, lactoperoxidase, lysozyme, and growth factors, and possess various antibacterial, antifungal, antiviral, anticancer, antioxidant, immunomodulatory properties, etc. This review describes the physico-chemical properties of milk and colostrum proteins and the natural functions they perform in the body and compares their composition between animal species (cows, goats, and sheep). The milk- and colostrum-based products can be used in dietary supplementation and for performing immunomodulatory functions; they can enhance the effects of certain drugs and can have a lethal effect on pathogenic microorganisms. Milk products are widely used in the treatment of dermatological diseases for promoting the healing of chronic wounds, hastening tissue regeneration, and the treatment of acne vulgaris or plaque psoriasis. They are also increasingly regarded as active ingredients that can improve the condition of the skin by reducing the number of acne lesions and blackheads, regulating sebum secretion, ameliorating inflammatory changes as well as bestowing a range of moisturizing, protective, toning, smoothing, anti-irritation, whitening, soothing, and antiaging effects.

Nicking and fragmentation are responsible for α-lactalbumin amyloid fibril formation at acidic pH and elevated temperature.

Amyloid fibrils are ordered aggregates that may be formed from disordered, partially unfolded, and fragments of proteins and peptides. There are several diseases, which are due to the formation and deposition of insoluble ß-sheet protein aggregates in various tissue, collectively known as amyloidosis. Here, we have used bovine α-lactalbumin as a model protein to understand the mechanism of amyloid fibril formation at pH 1.6 and 65°C under non-reducing conditions. Amyloid fibril formation is confirmed by Thioflavin T fluorescence and atomic force microscopy (AFM). Our finding demonstrates that hydrolysis of peptide bonds occurs under these conditions, which results in nicking and fragmentation. The nicking and fragmentation have been confirmed on non-reducing and reducing gel. We have identified the fragments by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The fragmentation may initiate nucleation as it coincides with AFM images. Conformational changes associated with monomer resulting in fibrillation are shown by circular dichroism and Raman spectroscopy. The current study highlights the importance of nicking and fragmentation in amyloid fibril formation, which may help understand the role of acidic pH and proteolysis under in vivo conditions in the initiation of amyloid fibril formation.