RESUMO
Enzymatic cross-linking is frequently used in bio-processing of dairy products since it could change the physiochemical and functional characterization. In our study, bovine α-lactalbumin was cross-linked by polyphenol oxidase from Agaricus bisporus and the changes in the structure, digestibility and allergenicity of α-lactalbumin were explored after cross-linking, and the structural alterations of the polymers were analyzed by circular dichroism spectroscopy, ultraviolet absorption spectroscopy and fluorescence spectroscopy. The digestibility of cross-linked α-lactalbumin was evaluated by simulated digestion in vitro. After that, the allergenicity of α-lactalbumin polymers was evaluated by detection of the specific IgE binding ability using an animal model. The results showed that the secondary and tertiary structures of various α-lactalbumin polymers exhibited a significant variation compared with those of untreated α-lactalbumin, and the cross-linked α-lactalbumin was relatively less susceptible to digestion. Moreover, the allergenicity of cross-linked polymers decreased significantly. These results suggested that there was a direct correlation between a loss of an α-helix and IgE binding to α-lactalbumin, which indicated that enzymatic cross-linking might be an efficient approach to reduce the allergenicity of bovine α-lactalbumin.
Assuntos
Alérgenos/química , Alérgenos/imunologia , Lactalbumina/química , Lactalbumina/imunologia , Agaricus/enzimologia , Alérgenos/genética , Animais , Sítios de Ligação , Catecol Oxidase/química , Bovinos , Feminino , Proteínas Fúngicas/química , Imunoglobulina E/química , Imunoglobulina E/imunologia , Lactalbumina/genética , Camundongos , Camundongos Endogâmicos BALB C , Polímeros/química , Estrutura Secundária de ProteínaRESUMO
This work studies α-lactalbumin adsorption on a charged substrate using Monte Carlo simulation. The protein is represented by a coarse-grained model with enough components as to reproduce the complex behavior of α-lactalbumin on electrically-charged substrates. The simulation results in particular can reproduce protein adsorption when both the protein and the substrate are negatively charged. The energetic and entropic contributions to the free energy of the adsorption process are estimated and analyzed. The effects of the charge regulation mechanism, the localization of titratable groups in α-lactalbumin as well as the distribution of small ions around the interface are studied in detail. Both the asymmetrical distribution of the charged groups of the protein and the counterion distribution play predominant roles in α-lactalbumin adsorption on a substrate with the same sign of electrical charge.
Assuntos
Simulação por Computador , Lactalbumina/química , Método de Monte Carlo , Polímeros/química , Adsorção , Humanos , Íons , Eletricidade Estática , Propriedades de SuperfícieRESUMO
A highly efficient method for aptamer screening with real-time monitoring of the SELEX process was described by silver decahedra nanoparticles (Ag10-NPs) enhanced surface plasmon resonance imaging (SPRI). A microarray chip was developed by immobilization of target protein (Lactoferrin (Lac)) and control proteins (α-lactalbumin (α), ß-lactoglobulin (ß), casein, and bovine serum albumin (BSA)) on the biochip surface. Ag10-NPs were conjugated with an ssDNA library (lib) (Ag10-NPs-library) that consisted of a central 40 nt randomized sequence and a 20 nt fixed primer sequence. Introduction of the Ag10-NPs-library to the SPRI flow channels drastically increased the sensitivity of SPRI signal for real-time monitoring of SELEX. The work allows rapid screening of potential targets, and yields nine aptamers with high affinity (nanomolar range) for Lac after only six-rounds of selection. The aptamer Lac 13-26 was then further tested by SPRI, and the results demonstrated that the aptamer had the capacity to be ultra-sensitive for specific detection of Lac. The novel SPRI-SELEX method demonstrated here showed many advantages of real-time evaluation, high throughput, and high efficiency.
Assuntos
Técnicas Biossensoriais , Lactoferrina/isolamento & purificação , Nanopartículas/química , Técnica de Seleção de Aptâmeros , Animais , Aptâmeros de Nucleotídeos/química , Caseínas/química , Bovinos , DNA de Cadeia Simples , Humanos , Lactalbumina/química , Lactoferrina/química , Análise Serial de Proteínas/métodos , Soroalbumina Bovina/química , Prata/química , Ressonância de Plasmônio de SuperfícieRESUMO
In downstream processing, the underlying adsorption mechanism of biomolecules to adsorbent material are still subject of extensive research. One approach to more mechanistic understanding is simulating this adsorption process and hereby the possibility to identify the parameters with strongest impact. So far this method was applied with all-atom molecular dynamics simulations of two model proteins on one cation exchanger. In this work we developed a molecular dynamics tool to simulate protein-adsorber interaction for various proteins on an anion exchanger and ran gradient elution experiments to relate the simulation results to experimental data. We were able to show that simulation results yield similar results as experimental data regarding retention behavior as well as binding orientation. We could identify arginines in case of cation exchangers and aspartic acids in case of anion exchangers as major contributors to binding.
Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Adsorção , Arginina/química , Ácido Aspártico/química , Cromatografia por Troca Iônica/métodos , Ácido Glutâmico/química , Lactalbumina/química , Luz , Método de Monte Carlo , Fosfolipases A2/química , Ribonuclease Pancreático/química , Espalhamento de Radiação , Sefarose/químicaRESUMO
The development of transgenic cloned animals offers new opportunities for agriculture, biomedicine and environmental science. Expressing recombinant proteins in dairy animals to alter their milk composition is considered beneficial for human health. However, relatively little is known about the expression profile of the proteins in milk derived from transgenic cloned animals. In this study, we compared the proteome and nutrient composition of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle that specifically express human α-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland with those from cloned non-transgenic (C) and conventionally bred normal animals (N). Protein expression profile identification was performed, 37 proteins were specifically expressed in the TC animals and 70 protein spots that were classified as 22 proteins with significantly altered expression levels in the TC and C groups compared to N group. Assessment of the relationship of the transgene effect and normal variability in the milk protein profiles in each group indicated that the variation in the endogenous protein profiles of the three TC groups was within the limit of natural variability. More than 50 parameters for the colostrum and mature milk were compared between each TC group and the N controls. The data revealed essentially similar profiles for all groups. This comprehensive study demonstrated that in TC cattle the mean values for the measured milk parameters were all within the normal range, suggesting that the expression of a transgene does not affect the composition of milk.
Assuntos
Animais Geneticamente Modificados , Leite/metabolismo , Animais , Bovinos , Cromatografia Líquida/métodos , Clonagem de Organismos , Eletroforese em Gel de Poliacrilamida , Feminino , Inocuidade dos Alimentos , Lactalbumina/química , Lactoferrina/química , Muramidase/química , Proteômica/métodos , Proteínas Recombinantes/química , Espectrometria de Massas em Tandem/métodos , TransgenesRESUMO
An economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (sCO(2)) as an acid to produce enriched fractions of α-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) from a commercial whey protein isolate (WPI) containing 20% α-LA and 55% ß-LG, through selective precipitation of α-LA. Pilot-scale experiments were performed around the optimal parameter range (T = 60 to 65 °C, P = 8 to 31 MPa, C = 5 to 15% (w/w) WPI) to quantify the recovery rates of the individual proteins and the compositions of both fractions as a function of processing conditions. Mass balances were calculated in a process flow-sheet to design a large-scale, semi-continuous process model using SuperproDesigner® software. Total startup and production costs were estimated as a function of processing parameters, product yield and purity. Temperature, T, pressure, P, and concentration, C, showed conflicting effects on equipment costs and the individual precipitation rates of the two proteins, affecting the quantity, quality, and production cost of the fractions considerably. The highest α-LA purity, 61%, with 80% α-LA recovery in the solid fraction, was obtained at T = 60 °C, C = 5% WPI, P = 8.3 MPa, with a production cost of $8.65 per kilogram of WPI treated. The most profitable conditions resulted in 57%-pure α-LA, with 71% α-LA recovery in the solid fraction and 89% ß-LG recovery in the soluble fraction, and production cost of $5.43 per kilogram of WPI treated at T = 62 °C, C = 10% WPI and P = 5.5 MPa. The two fractions are ready-to-use, new food ingredients with a pH of 6.7 and contain no residual acid or chemical contaminants.
Assuntos
Dióxido de Carbono/química , Cromatografia com Fluido Supercrítico/métodos , Proteínas do Leite/isolamento & purificação , Animais , Bovinos , Cromatografia com Fluido Supercrítico/economia , Concentração de Íons de Hidrogênio , Lactalbumina/química , Lactalbumina/isolamento & purificação , Lactoglobulinas/química , Lactoglobulinas/isolamento & purificação , Proteínas do Leite/química , Projetos Piloto , Pressão , Temperatura , Proteínas do Soro do LeiteRESUMO
The salt and pH dependent association of hen egg white lysozyme with alpha-lactalbumin whey proteins has been studied using molecular level Monte Carlo simulations. A highly uneven charge distribution of alpha-lactalbumin leads to strongly ordered heterodimers that may facilitate the formation of structured, mesoscopic aggregates. This electrostatic steering gives rise to 80% alignment at 5 mM 1 : 1 salt which, due to screening, diminishes to 60% at 100 mM salt. The free energy of interaction minima, dominated by electrostatics, ranges between -9 kT at 1 mM salt to -2 kT at 100 mM (neutral pH). Calculated osmotic second virial cross coefficients indicate complexation in the pH interval 6-10. Multivalent ions are found to effectively destabilize the protein complex and, at constant ionic strength, the order is La(3+) > Ca(2+) > Mg(2+) > Na(+). Upon binding of calcium to alpha-lactalbumin both the interaction and orientational alignment with lysozyme are reduced due to induced changes in the whey protein charge distribution. This potentially explains the experimentally observed absence of supramolecular structuring for the calcium loaded holo alpha-lactalbumin. Where available, good agreement is found with experimental data.
Assuntos
Lactalbumina/química , Muramidase/química , Eletricidade Estática , Animais , Galinhas , Dimerização , Concentração de Íons de Hidrogênio , Método de Monte Carlo , Muramidase/metabolismo , Sais/químicaRESUMO
The degree of irreversible aggregation and the aggregation velocity constant of alpha-lactalbumin (alpha-la) were determined by three methods based on different principles: low-intensity ultrasound as a novel method for this purpose, DSC, and HPLC. The denaturation process of alpha-la causes a decrease in the ultrasonic velocity due to the conformation change of alpha-la molecules. This decrease is a function of the concentration of native alpha-la in the sample. A linear correlation was found between the degree of aggregation of alpha-la determined by these three methods. There is no significant difference between the aggregation velocity constants determined by the three methods. The results show that the ultrasonic method is capable of quantifying the degree of aggregation of a protein, offering an alternative method.
Assuntos
Temperatura Alta , Lactalbumina/química , Desnaturação Proteica , Animais , Varredura Diferencial de Calorimetria , Bovinos , Cromatografia Líquida de Alta Pressão , Cinética , UltrassomRESUMO
Both natural and synthetic polyelectrolytes form strong complexes with a variety of proteins. One peculiar phenomenon is that association can take place even when the protein and the polyelectrolyte carry the same charge. This has been interpreted as if the ion-dipole interaction can overcome the repulsive ion-ion interaction. On the basis of Monte Carlo simulations and perturbation theory, we propose a different explanation for the association, namely, charge regulation. We have investigated three different protein-polymer complexes and found that the induced ionization of amino acid residues due to the polyelectrolyte leads to a surprisingly strong attractive interaction between the protein and the polymer. The extra attraction from this charge-induced charge interaction can be several kT and is for the three cases studied here, lysozyme, alpha-lactalbumin, and beta-lactoglobulin, of the same magnitude or stronger than the ion-dipole interaction. The magnitude of the induced charge is governed by a response function, the protein charge capacitance Z2-Z2. This fluctuation term can easily be calculated in a simulation or measured in a titration experiment.
Assuntos
Eletrólitos/química , Modelos Químicos , Polímeros/química , Proteínas/química , Simulação por Computador , Concentração de Íons de Hidrogênio , Lactalbumina/química , Lactoglobulinas/química , Método de Monte Carlo , Muramidase/química , Eletricidade EstáticaRESUMO
In structural proteomics, it is necessary to efficiently screen in a high-throughput manner for the presence of stable structures in proteins that can be subjected to subsequent structure determination by X-ray or NMR spectroscopy. Here we illustrate that the (1)H chemical distribution in a protein as detected by (1)H NMR spectroscopy can be used to probe protein structural stability (e.g., the presence of stable protein structures) of proteins in solution. Based on experimental data obtained on well-structured proteins and proteins that exist in a molten globule state or a partially folded alpha-helical state, a well-defined threshold exists that can be used as a quantitative benchmark for protein structural stability (e.g., foldedness) in solution. Additionally, in this chapter we describe a largely automated strategy for rapid fold validation and structure-based backbone signal assignment. Our methodology is based on a limited number of NMR experiments (e.g., HNCA and 3D NOESY-HSQC) and performs a Monte Carlo-type optimization. The novel feature of the method is the opportunity to screen for structural fragments (e.g., template scanning). The performance of this new validation tool is demonstrated with applications to a diverse set of proteins.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Dobramento de Proteína , Estrutura Terciária de Proteína , Cálcio/metabolismo , Interpretação Estatística de Dados , Estabilidade Enzimática , Lactalbumina/química , Lactalbumina/metabolismo , Método de Monte CarloRESUMO
Electrostatic complexation of flexible polyanions with the whey proteins alpha-lactalbumin and beta-lactoglobulin is studied using Monte Carlo simulations. The proteins are considered at their respective isoelectric points. Discrete charges on the model polyelectrolytes and proteins interact through Debye-Huckel potentials. Protein excluded volume is taken into account through a coarse-grained model of the protein shape. Consistent with experimental results, it is found that alpha-lactalbumin complexes much more strongly than beta-lactoglobulin. For alpha-lactalbumin, strong complexation is due to localized binding to a single large positive "charge patch," whereas for beta-lactoglobulin, weak complexation is due to diffuse binding to multiple smaller charge patches.
Assuntos
Lactalbumina/química , Lactoglobulinas/química , Proteínas do Leite/química , Modelos Químicos , Modelos Moleculares , Complexos Multiproteicos/química , Ânions , Sítios de Ligação , Simulação por Computador , Dimerização , Elasticidade , Eletroquímica/métodos , Método de Monte Carlo , Ligação ProteicaRESUMO
We present a method for determining the ensembles of native protein structures that result from the large fluctuations of low probability revealed by hydrogen-exchange experiments. The measured protection factors are used to bias Monte Carlo simulations to sample the structures of the exchange competent species. The approach is illustrated by its application to the case of alpha-lactalbumin.
Assuntos
Hidrogênio/química , Proteínas/química , Humanos , Hidrogênio/metabolismo , Ligação de Hidrogênio , Cinética , Lactalbumina/química , Lactalbumina/metabolismo , Modelos Químicos , Método de Monte Carlo , Estrutura Secundária de Proteína , Proteínas/metabolismoRESUMO
The ability of proteins to fold to well defined compact structures is one of the most remarkable examples of the effect of natural selection on biological molecules. To understand their properties, including the stability, the mechanism of folding, and the possibilities of misfolding and association, it is necessary to know the protein free energy landscape. We use NMR data as restraints in a Monte Carlo sampling procedure to determine the ensemble of structures populated by human alpha-lactalbumin in the presence of increasing concentrations of urea. The ensembles of structures that represent the partially folded states of the protein show that two structural cores, corresponding to portions of the alpha and beta domains of the native protein, are preserved even when the native-like interactions that define their existence are substantially weakened. Analysis of the network of residual contacts reveals the presence of a complex interface region between the two structural cores and indicates that the development of specific interactions within this interface is the key step in achieving the native structure. The relative probabilities of the conformations determined from the NMR data are used to construct a coarse-grained free energy landscape for alpha-lactalbumin in the absence of urea. The form of the landscape, together with the existence of distinct cores, supports the concept that robustness and modularity are the properties that make possible the folding of complex proteins.
Assuntos
Dobramento de Proteína , Humanos , Concentração de Íons de Hidrogênio , Lactalbumina/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Método de Monte Carlo , Conformação Proteica , Estrutura Terciária de Proteína , Termodinâmica , Ureia/químicaRESUMO
A diagnostic for assessing the quality of a fold has been developed to which further criteria can be progressively added. The goal is to create a measure that can follow the status of a protein structure in a simulation or modeling process, when the answer (the experimental structure) is not known in advance, rather than simply reject deliberate misfolds. This places greater emphasis on the need to study, and calibrate against, marginal cases, i.e., unusual native structures, incomplete structures, partially erroneous X-ray structures, good models, poor models, and the effect of cofactors. The first three terms introduced in the diagnostic are appropriate core-forming properties or noncore properties of residues in relation to tertiary structure, appropriate neighboring structure density for each residue in relation to tertiary structure, and secondary structure consistency. While the method emerges as a useful simulation analysis tool, we find a need for further fine-tuning to diminish sensitivity to minor conformational changes that retain essential features of the fold, balanced against the need to obtain a more sensitive response when a conformational change involves less physically meaningful interatomic interactions. This dual utility is difficult to obtain: the investigation highlights some of the issues. Initial attempts to obtain it have led to terms in the diagnostic that are admittedly complex: simplifications must also be explored.
Assuntos
Proteínas de Bactérias , Modelos Moleculares , Peptídeos , Dobramento de Proteína , Estrutura Terciária de Proteína , Animais , Antibacterianos/química , Humanos , Lactalbumina/química , Domínios de Homologia de srcRESUMO
A number of proteins and peptides have been incubated with some Immobiline chemicals commonly used in the production of immobilised pH gradients for isoelectric focusing. After various incubation intervals, the resulting reaction mixtures were examined by matrix-assisted laser desorption/ionisation mass spectrometry. At pH 9-10, and after 15-h incubation time, no significant interaction was observed with the two of the investigated proteins which have no Cys residues in their sequences. On the other hand, intense multiple reaction channels were observed with sequences containing a number of Cys residues. The present measurements provide useful information on the kinetics of the reaction and its sensitivity to both the pK(a) of the Immobiline chemicals and the presence of Cys in the investigated sequences. Post source decay measurements on peptides with and without Cys in their sequences provided unambiguous evidence for the involvement of this residue in the reaction conducted at pH 9-10. Possible implications of some of the present deductions for isoelectric focusing separations on immobilised pH gradients are discussed.