RESUMEN
Enzymes are conformationally dynamic, and their dynamical properties play an important role in regulating their specificity and evolvability. In this context, substantial attention has been paid to the role of ligand-gated conformational changes in enzyme catalysis; however, such studies have focused on tremendously proficient enzymes such as triosephosphate isomerase and orotidine 5'-monophosphate decarboxylase, where the rapid (µs timescale) motion of a single loop dominates the transition between catalytically inactive and active conformations. In contrast, the (ßα)8-barrels of tryptophan and histidine biosynthesis, such as the specialist isomerase enzymes HisA and TrpF, and the bifunctional isomerase PriA, are decorated by multiple long loops that undergo conformational transitions on the ms (or slower) timescale. Studying the interdependent motions of multiple slow loops, and their role in catalysis, poses a significant computational challenge. This work combines conventional and enhanced molecular dynamics simulations with empirical valence bond simulations to provide rich details of the conformational behavior of the catalytic loops in HisA, PriA, and TrpF, and the role of their plasticity in facilitating bifunctionality in PriA and evolved HisA variants. In addition, we demonstrate that, similar to other enzymes activated by ligand-gated conformational changes, loops 3 and 4 of HisA and PriA act as gripper loops, facilitating the isomerization of the large bulky substrate ProFAR, albeit now on much slower timescales. This hints at convergent evolution on these different (ßα)8-barrel scaffolds. Finally, our work reemphasizes the potential of engineering loop dynamics as a tool to artificially manipulate the catalytic repertoire of TIM-barrel proteins.
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The cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. An in-depth understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical, and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for the cooperative interplay between the nucleophilic and general-acid catalytic groups in the wild-type enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is â¼104-fold. Cooperativity with the catalytic acid adds ≥102-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.
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Active nanophotonics can be realized by controlling the optical properties of materials with external magnetic fields. Here, we explore the influence of optical anisotropy on the magneto-optical activity in nonmagnetic hyperbolic nanoparticles. We demonstrate that the magneto-optical response is driven by the hyperbolic dispersion via the coupling of metallic-induced electric and dielectric-induced magnetic dipolar optical modes with static magnetic fields. Magnetic circular dichroism experiments confirm the theoretical predictions and reveal tunable magneto-optical activity across the visible and near infrared spectral range.
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DNA-binding proteins play an important role in gene regulation and cellular function. The transcription factors MarA and Rob are two homologous members of the AraC/XylS family that regulate multidrug resistance. They share a common DNA-binding domain, and Rob possesses an additional C-terminal domain that permits binding of low-molecular weight effectors. Both proteins possess two helix-turn-helix (HTH) motifs capable of binding DNA; however, while MarA interacts with its promoter through both HTH-motifs, prior studies indicate that Rob binding to DNA via a single HTH-motif is sufficient for tight binding. In the present work, we perform microsecond time scale all-atom simulations of the binding of both transcription factors to different DNA sequences to understand the determinants of DNA recognition and binding. Our simulations characterize sequence-dependent changes in dynamical behavior upon DNA binding, showcasing the role of Arg40 of the N-terminal HTH-motif in allowing for specific tight binding. Finally, our simulations demonstrate that an acidic C-terminal loop of Rob can control the DNA binding mode, facilitating interconversion between the distinct DNA binding modes observed in MarA and Rob. In doing so, we provide detailed molecular insight into DNA binding and recognition by these proteins, which in turn is an important step toward the efficient design of antivirulence agents that target these proteins.
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Proteínas de Unión al ADN , Proteínas de Escherichia coli , Factores de Transcripción , Proteínas Bacterianas , Sitios de Unión , ADN/genética , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Secuencias Hélice-Giro-Hélice , Transactivadores/metabolismo , Factores de Transcripción/genéticaRESUMEN
We introduce a novel concept of hybrid metal-dielectric meta-antenna supporting type II hyperbolic dispersion, which enables full control of absorption and scattering of light in the visible/near-infrared spectral range. This ability lies in the different nature of the localized hyperbolic Bloch-like modes excited within the meta-antenna. The experimental evidence is corroborated by a comprehensive theoretical study. In particular, we demonstrate that two main modes, one radiative and one non-radiative, can be excited by direct coupling with the free-space radiation. We show that the scattering is the dominating electromagnetic decay channel, when an electric dipolar mode is induced in the system, whereas a strong absorption process occurs when a magnetic dipole is excited. Also, by varying the geometry of the system, the relative ratio of scattering and absorption, as well as their relative enhancement and/or quenching, can be tuned at will over a broad spectral range, thus enabling full control of the two channels. Importantly, both radiative and nonradiative modes supported by our architecture can be excited directly with far-field radiation. This is observed to occur even when the radiative channels (scattering) are almost totally suppressed, thereby making the proposed architecture suitable for practical applications. Finally, the hyperbolic meta-antennas possess both angular and polarization independent structural integrity, unlocking promising applications as hybrid meta-surfaces or as solvable nanostructures.
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Although gold nanoparticles (AuNPs) are currently used in several industrial products and biomedical applications, information about their biological effects is very limited. Thus, it is becoming crucial to assess their safety and adequately investigate the complexity of cell-nanoparticles interactions. In this work, the Balb/3T3 mouse fibroblast cell line was selected as an in vitro model to study the effects of AuNPs. Alteration of cellular processes and biochemical pathways caused by AuNPs exposure was investigated by analysing the differentially expressed proteome. Of interest was the difference observed in the protein pattern expression of cells exposed to AuNPs. It was found that 88 and 83 proteins were de-regulated after exposure to 5 and 15nm AuNPs, respectively. Analysis of the proteome revealed that AuNPs triggers several pathways related to cellular growth and proliferation, cell morphology, cell cycle regulation, cellular function and maintenance, oxidative stress, and inflammatory response. Moreover, SPR analysis showed an increase of ECM proteins biosynthesis in cells exposed to AuNPs. We observed by TEM analysis that NPs are internalized and confined mainly in autophagosomes. Endoplasmic reticulum stressed and modification at mitochondrial level occurred. This study aims to improve existing knowledge necessary for a correct assessment of the balance between AuNPs potential adverse and beneficial effects and might have important implications for biomedical applications (e.g. nanomedicine). To conclude proteomics link to system biology analysis is a valuable tool to understand and predict nanoparticles' toxicity, furthermore it has the potential to reveal pathways that may not be immediately evident with classical toxicological assays.
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Oro/toxicidad , Nanopartículas del Metal/toxicidad , Proteómica/métodos , Animales , Células 3T3 BALB , Fraccionamiento Celular , Cromatografía Líquida de Alta Presión , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Fibronectinas/biosíntesis , Fibronectinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hidrólisis , Procesamiento de Imagen Asistido por Computador , Espectrometría de Masas , Ratones , Microscopía Electrónica de Transmisión , Péptidos/química , Fosforilación , Transducción de Señal/efectos de los fármacos , Resonancia por Plasmón de SuperficieRESUMEN
Continuous 295 nm excitation of whey protein bovine apo-α-lactalbumin (apo-bLA) results in an increase of tryptophan fluorescence emission intensity, in a progressive red-shift of tryptophan fluorescence emission, and breakage of disulphide bridges (SS), yielding free thiol groups. The increase in fluorescence emission intensity upon continuous UV-excitation is correlated with the increase in concentration of free thiol groups in apo-bLA. UV-excitation and consequent SS breakage induce conformational changes on apo-bLA molecules, which after prolonged illumination display molten globule spectral features. The rate of tryptophan fluorescence emission intensity increase at 340 nm with excitation time increases with temperature in the interval 9.3-29.9°C. The temperature-dependent 340 nm emission kinetic traces were fitted by a 1st order reaction model. Native apo-bLA molecules with intact SS bonds and low tryptophan emission intensity are gradually converted upon excitation into apo-bLA molecules with disrupted SS, molten-globule-like conformation, high tryptophan emission intensity and red-shifted tryptophan emission. Experimental Ahrrenius activation energy was 21.8 ± 2.3 kJ x mol(-1). Data suggests that tryptophan photoionization from the S(1) state is the likely pathway leading to photolysis of SS in apo-bLA. Photoionization mechanism(s) of tryptophan in proteins and in solution and the activation energy of tryptophan photoionization from S(1) leading to SS disruption in proteins are discussed. The observations present in this paper raise concern regarding UV-light pasteurization of milk products. Though UV-light pasteurization is a faster and cheaper method than traditional thermal denaturation, it may also lead to loss of structure and functionality of milk proteins.
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Apoproteínas/química , Disulfuros/química , Lactalbúmina/química , Procesos Fotoquímicos , Rayos Ultravioleta , Animales , Bovinos , Modelos Moleculares , Conformación Proteica/efectos de la radiación , Espectrometría de Fluorescencia , Compuestos de Sulfhidrilo/química , TemperaturaRESUMEN
Medical interest in nanotechnology originates from a belief that nanoscale therapeutic devices can be constructed and directed towards its target inside the human body. Such nanodevices can be engineered by coupling superparamagnetic nanoparticle to biomedically active proteins. We hereby report the immobilization of a PhEst, a S-formylglutathione hydrolase from the psychrophilic P. haloplanktis TAC125 onto the gold coated surface of modified superparamagnetic core-shell nanoparticles (Fe(3)O(4)@Au). The synthesis of the nanoparticles is also reported. S-formylglutathione hydrolases constitute a family of ubiquitous enzymes which play a key role in formaldehyde detoxification both in prokaryotes and eukaryotes. PhEst was originally annotated as a putative feruloyl esterase, an enzyme that releases ferulic acid (an antioxidant reactive towards free radicals such as reactive oxygen species) from polysaccharides esters. Dynamic light scattering, scanning electron microscopy with energy dispersive X-ray spectroscopy, UV-visible absorption spectroscopy, fluorescence spectroscopy, magnetic separation technique and enzyme catalytic assay confirmed the chemical composition of the gold covered superparamagnetic nanoparticles, the binding and activity of the enzyme onto the nanoparticles. Activity data in U/ml confirmed that the immobilized enzyme is approximately 2 times more active than the free enzyme in solution. Such particles can be directed with external magnetic fields for bio-separation and focused towards a medical target for therapeutical as well as bio-sensor applications.
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Equipos y Suministros , Magnetismo , Nanopartículas de Magnetita/química , Nanotecnología/instrumentación , Análisis Espectral , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Oro/química , Modelos Moleculares , Conformación Proteica , Pseudomonas/enzimología , Propiedades de Superficie , Tioléster Hidrolasas/química , Tioléster Hidrolasas/metabolismoRESUMEN
Light assisted molecular immobilization has been used for the first time to engineer covalent bioconjugates of superparamagnetic nanoparticles and proteins. The technology involves disulfide bridge disruption upon UV excitation of nearby aromatic residues. The close spatial proximity of aromatic residues and disulfide bridges is a conserved structural feature in proteins. The created thiol groups bind thiol reactive surfaces leading to oriented covalent protein immobilization. We have immobilized a model carrier protein, bovine serum albumin, onto Fe(3)O(4)@Au core-shell nanoparticles as well as arrayed it onto optically flat thiol reactive surfaces. This new immobilization technology allows for ultra high dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalized active new biosensor materials, biomarkers identification and the development of nanoparticles based novel drug delivery system.
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Fotones , Albúmina Sérica Bovina/química , Secuencia de Aminoácidos , Microscopía Electrónica de Rastreo , Modelos Moleculares , Datos de Secuencia Molecular , Nanopartículas , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Rayos UltravioletaRESUMEN
We developed a method for immobilization of biomolecules onto thiol functionalized surfaces according to UV diffraction patterns. UV light-assisted molecular immobilization proceeds through the formation of free, reactive thiol groups that can bind covalently to thiol reactive surfaces. We demonstrate that, by shaping the pattern of the UV light used to induce molecular immobilization, one can control the pattern of immobilized molecules onto the surface. Using a single-aperture spatial mask, combined with the Fourier transforming property of a focusing lens, we show that submicrometer (0.7 µm) resolved patterns of immobilized prostate-specific antigen biomolecules can be created. If a dual-aperture spatial mask is used, the results differ from the expected Fourier transform pattern of the mask. It appears as a superposition of two diffraction patterns produced by the two apertures, with a fine structured interference pattern superimposed.
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Procesos Fotoquímicos , Antígeno Prostático Específico/efectos de la radiación , Rayos Ultravioleta , Análisis de Fourier , Proteínas Inmovilizadas , Lentes , Compuestos de Sulfhidrilo/farmacología , Propiedades de Superficie/efectos de los fármacosRESUMEN
We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti-PSA biosensor arrays using UV light-assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti-PSA in solution and immobilized Fab anti-PSA cross-reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems.
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Biomarcadores de Tumor/inmunología , Técnicas Biosensibles/métodos , Proteínas Inmovilizadas/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Antígeno Prostático Específico/inmunología , Neoplasias de la Próstata/diagnóstico , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/química , Humanos , Proteínas Inmovilizadas/análisis , Proteínas Inmovilizadas/química , Inmunoensayo/métodos , Fragmentos Fab de Inmunoglobulinas/química , Masculino , Microscopía Fluorescente , Modelos Moleculares , Antígeno Prostático Específico/análisis , Antígeno Prostático Específico/química , Análisis por Matrices de Proteínas/métodos , Espectrometría de Fluorescencia , Rayos UltravioletaRESUMEN
S-formylglutathione hydrolases (FGHs) constitute a family of ubiquitous enzymes which play a key role in formaldehyde detoxification both in prokaryotes and eukaryotes, catalyzing the hydrolysis of S-formylglutathione to formic acid and glutathione. While a large number of functional studies have been reported on these enzymes, few structural studies have so far been carried out. In this article we report on the functional and structural characterization of PhEst, a FGH isolated from the psychrophilic bacterium Pseudoalteromonas haloplanktis. According to our functional studies, this enzyme is able to efficiently hydrolyze several thioester substrates with very small acyl moieties. By contrast, the enzyme shows no activity toward substrates with bulky acyl groups. These data are in line with structural studies which highlight for this enzyme a very narrow acyl-binding pocket in a typical alpha/beta-hydrolase fold. PhEst represents the first cold-adapted FGH structurally characterized to date; comparison with its mesophilic counterparts of known three-dimensional structure allowed to obtain useful insights into molecular determinants responsible for the ability of this psychrophilic enzyme to work at low temperature.
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Pseudoalteromonas/enzimología , Tioléster Hidrolasas/química , Secuencia de Aminoácidos , Biotransformación , Dominio Catalítico , Frío , Cristalografía por Rayos X , Dimerización , Estabilidad de Enzimas , Formaldehído/metabolismo , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Cuaternaria de Proteína , Pseudoalteromonas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismoRESUMEN
Psychrophiles produce cold-evolved enzymes that display a high catalytic efficiency, associated with a low thermal stability. In recent years, these enzymes have attracted the attention of scientists because of their peculiar properties that render them particularly useful in investigating the relationship existing between enzyme stability and flexibility on one hand, and enzyme activity on the other hand. Among these enzymes, the esterases, and particularly the feruloyl esterases, have potential uses over a broad range of applications in the agro-food industries. In recent years, the number of microbial feruloyl esterase activities has increased in the growing genome databases. Based on substrate utilization data and supported by primary sequence identity, four subclasses of esterase have been characterized so far. Up to the present, ten genomes from psychrophilic bacteria have been completely sequenced and additional fourteen genomes are under investigation. From the bacteria strains whose genome has been completely sequenced, we analyzed the presence of esterase genes, both the putative genes and the determined experimentally genes, and performed a ClustalW analysis for feruloyl esterases. Major details will be presented for the ORF PSHAa1385 from P. haloplanktis TAC125 that recently has been studied in our research group. In addition, the potential biotechnology applications of this class of enzymes will be discussed.
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Adaptación Fisiológica , Hidrolasas de Éster Carboxílico/fisiología , Frío , Pseudoalteromonas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas , Catálisis , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/fisiología , Análisis de Secuencia de ProteínaRESUMEN
The effects of hydrostatic pressure on the structure and stability of porcine odorant-binding protein (pOBP) in the presence and absence of the odorant molecule 2-isobutyl-3-methoxypyrazine (IBMP) were studied by steady-state and time-resolved fluorescence spectroscopy as well as by molecular dynamics simulation. The authors found that the application of moderate values of hydrostatic pressure to pOBP solutions perturbed the microenvironment of Trp(16) and disrupted its highly quenched complex with Met(39). In addition, compared with the protein in the absence of IBMP, the MD simulations experiments carried out at different pressures highlighted the role of this ligand in stabilizing the Trp(16)/Met(39) interaction even at 2000 bar. The obtained results will assist for the tailoring of this protein as specific sensing element in a new class of fluorescence-based biosensors for the detection of explosives.
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Receptores Odorantes/química , Animales , Simulación por Computador , Modelos Moleculares , Estructura Molecular , Presión , Pirazinas/química , Espectrometría de Fluorescencia , Porcinos , Factores de TiempoRESUMEN
The complete genome of the psychrophilic bacteria Pseudoalteromonas haloplanktis TAC 125, recently published, owns a gene coding for a putative esterase activity corresponding to the ORF PSHAa1385, also classified in the Carbohydrate Active Enzymes database (CAZY) belonging to family 1 of carbohydrate esterase proteins. This ORF is 843 bp in length and codes for a protein of 280 amino acid residues. In this study we characterized and cloned the PSHAa1385 gene in Escherichia coli. We also characterized the recombinant protein by biochemical and biophysical methodologies. The PSHAa1385 gene sequence showed a significant homology with several carboxyl-esterase and acetyl-esterase genes from gamma-proteobacteria genera and yeast. The recombinant protein exhibited a significant activity towards pNP-acetate, alpha-and beta-naphthyl acetate as generic substrates, and 4-methylumbelliferyl p-trimethylammonio cinnamate chloride (MUTMAC) as a specific substrate, indicating that the protein exhibits a feruloyl esterase activity that it is displayed by similar enzymes present in other organisms. Finally, a three-dimensional model of the protein was built and the amino acid residues involved in the catalytic function of the protein were identified.
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Hidrolasas de Éster Carboxílico/genética , Genes Bacterianos , Pseudoalteromonas/enzimología , Pseudoalteromonas/genética , Adaptación Fisiológica , Secuencia de Aminoácidos , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Clonación Molecular , Frío , Estabilidad de Enzimas , Esterasas/genética , Gammaproteobacteria/enzimología , Gammaproteobacteria/genética , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Especificidad de la Especie , TermodinámicaRESUMEN
Patulin is a toxic secondary metabolite of a number of fungal species belonging to the genera Penicillum and Aspergillus. It has been mainly isolated from apples and apple products contaminated with the common storage-rot fungus of apples, Penicillum expansum, but it has also been extracted from rotten fruits, moldy feeds, and stored cheese. Human exposure to patulin can lead to serious health problems, and according to a long-term investigation in rats, the World Health Organization has set a tolerable weekly intake of 7 ppb body weight. The content of patulin in foods has been restricted to 50 ppb in many countries. Conventional analytical detection methods involve chromatographic analyses, such as HPLC, GC, and, more recently, techniques such as LC/MS and GC/MS. However, extensive protocols of sample cleanup are required prior to the analysis, and to accomplish it, expensive analytical instrumentation is necessary. An immunochemical analytical method, based on highly specific antigen-antibody interactions, would be desirable, offering several advantages compared to conventional techniques, i.e., low cost per sample, high selectivity, high sensitivity, and high throughput. In this paper, the synthesis of two new derivatives of patulin is described, along with their conjugation to the bovine serum albumin for the production of polyclonal antibodies. Finally, a fluorescence competitive immunoassay was developed for the on-line detection of patulin.
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Técnica del Anticuerpo Fluorescente/métodos , Análisis de los Alimentos/métodos , Contaminación de Alimentos , Patulina/análisis , Animales , Anticuerpos/química , Anticuerpos/inmunología , Unión Competitiva , Western Blotting , Bovinos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Patulina/análogos & derivados , Patulina/síntesis química , Patulina/química , Patulina/inmunología , Conejos , Albúmina Sérica Bovina/químicaRESUMEN
The effect of the pressure on the structure and stability of the D-galactose/D-glucose binding protein from Escherichia coli in the absence (GGBP) and in the presence (GGBP/Glc) of glucose was studied by Fourier transform infrared (FT-IR) spectroscopy and molecular dynamic (MD) simulations. FT-IR spectroscopy experiments showed that the protein beta-structures are more resistant than alpha-helices structures to pressure value increases. In addition, the infrared data indicated that the binding of glucose stabilizes the protein structure against high pressure values, and the protein structure does not completely unfold up to pressure values close to 9000 bar. MD simulations allow a prediction of the most probable configuration of the protein, consistent with the increasing pressures on the two systems. The detailed analysis of the structures at molecular level confirms that, among secondary structures, alpha-helices are more sensitive than beta-structures to the destabilizing effect of high pressure and that glucose is able to preserve the structure of the protein in the complex. Moreover, the evidence of the different resistance of the two domains of this protein to high pressure is investigated and explained at a molecular level, indicating the importance of aromatic amino acid in protein stabilization.
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Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Transporte de Monosacáridos/química , Pliegue de Proteína , Galactosa/química , Glucosa/química , Presión , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
In this work is presented the first attempt to develop a fluorescence assay for detection of traces of gluten in food by utilizing the recombinant glutamine-binding protein (GlnBP) from E. coli. We found that GlnBP specifically binds the sequence of amino acids present both in gliadin and other prolamines classified as toxic for celiac patients. Affinity chromatography experiments together with mass spectrometry experiments demonstrated that GlnBP can bind the following amino acid sequence XXQPQPQQQQQQQQQQQQL. Sequence alignment experiments pointed out that this sequence is exclusively representative of the gliadin and the other prolamines considered toxic for celiac patients. These findings suggest the development of a competitive resonance energy transfer (RET) assay for an easy and rapid detection of this sequence in raw and cooked food.
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Proteínas Portadoras/metabolismo , Escherichia coli/genética , Análisis de los Alimentos/métodos , Gliadina/aislamiento & purificación , Unión Proteica , Triticum/genética , Secuencia de Aminoácidos , Cromatografía de Afinidad , Gliadina/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Alineación de Secuencia , Espectrometría de FluorescenciaRESUMEN
In this report we show the purification to homogeneity and a partial characterization of a new odorant-binding protein from Canis familiar (CfOBP) nasal mucosa. In addition, we report preliminary data on the utilization of CfOBP as a probe for the development of a refractive index-based biosensor.