RESUMO
STD NMR spectroscopy is a powerful ligand-observed NMR tool for screening and characterizing the interactions of small molecules and low molecular weight fragments with a given macromolecule, identifying the main intermolecular contacts in the bound state. It is also a powerful analytical technique for the accurate determination of protein-ligand dissociation constants (KD) of medium-to-weak affinity, of interest in the pharmaceutical industry. However, accurate KD determination and epitope mapping requires a long series of experiments at increasing saturation times to carry out a full analysis using the so-called STD NMR build-up curve approach and apply the "initial slopes approximation". Here, we have developed a new protocol to bypass this important limitation, which allows us to obtain initial slopes by using just two saturation times and, hence, to very quickly determine precise protein-ligand dissociation constants by STD NMR.
Assuntos
Imageamento por Ressonância Magnética , Proteínas , Ligantes , Proteínas/química , Espectroscopia de Ressonância Magnética/métodos , Mapeamento de Epitopos , Ligação ProteicaRESUMO
The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-ß-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.
Assuntos
Clostridiales/metabolismo , Mucina-1/metabolismo , Sistema ABO de Grupos Sanguíneos/imunologia , Antígenos de Grupos Sanguíneos/imunologia , Clostridiales/genética , Clostridiales/fisiologia , Microbioma Gastrointestinal , Trato Gastrointestinal , Glicosídeo Hidrolases/metabolismo , Humanos , Mucinas/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Ruminococcus/genética , Ruminococcus/metabolismo , Especificidade por Substrato , Espectrometria de Massas em Tandem/métodosRESUMO
The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.
Assuntos
Antitrombinas , Heparina , Oligossacarídeos , Humanos , Antitrombinas/química , Antitrombinas/metabolismo , Sítios de Ligação , Mapeamento de Epitopos/métodos , Heparina/química , Heparina/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Ligação Proteica , Solventes/químicaRESUMO
We have combined saturation transfer difference NMR (STD NMR) with chemical shift imaging (CSI) and controlled concentration gradients of small molecule ligands to develop imaging STD NMR, a new tool for the assessment of protein-ligand interactions. Our methodology allows the determination of protein-ligand dissociation constants (KD) and assessment of the binding specificity in a single NMR tube, avoiding time-consuming titrations. We demonstrate the formation of suitable and reproducible concentration gradients of ligand along the vertical axis of the tube, against homogeneous protein concentration, and present a CSI pulse sequence for the acquisition of STD NMR experiments at different positions along the sample tube. Compared to the conventional methodology in which the [ligand]/[protein] ratio is increased manually, we can perform STD NMR experiments at a greater number of ratios and construct binding epitopes in a fraction (â¼20%) of the experimental time. Second, imaging STD NMR also allows us to screen for non-specific binders, by monitoring any variation of the binding epitope map at increasing [ligand]/[protein] ratios. Hence, the proposed method does carry the potential to speed up and smooth out the drug discovery process.
Assuntos
Imageamento por Ressonância Magnética , Proteínas , Sítios de Ligação , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Ligação Proteica , Epitopos/químicaRESUMO
Lectins are capable of reading out the structural information contained in carbohydrates through specific recognition processes. Determining the binding epitope of the sugar is fundamental to understanding this recognition event. Nuclear magnetic resonance (NMR) is a powerful tool to obtain this structural information in solution; however, when the sugar involved is a complex oligosaccharide, such as high mannose, the signal overlap found in the NMR spectra precludes an accurate analysis of the interaction. The introduction of tags into these complex oligosaccharides could overcome these problems and facilitate NMR studies. Here, we show the preparation of the Man9 of high mannose with some fluorine tags and the study of the interaction with its receptor, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). This fluorinated ligand has allowed us to apply heteronuclear two-dimensional (2D) 1H,19F STD-TOCSYreF NMR experiments, using the initial slope approach, which has facilitated the analysis of the Man9/DC-SIGN interaction, unequivocally providing the binding epitope.
Assuntos
Lectinas Tipo C , Manose , Humanos , Manose/química , Lectinas Tipo C/metabolismo , Oligossacarídeos/química , Açúcares , Espectroscopia de Ressonância Magnética , Epitopos , Células DendríticasRESUMO
BACKGROUND AND AIMS: Arbuscular mycorrhizal (AM) fungi enhance the uptake of water and minerals by the plant hosts, alleviating plant stress. Therefore, AM fungal-plant interactions are particularly important in drylands and other stressful ecosystems. We aimed to determine the combined and independent effects of above- and below-ground plant community attributes (i.e. diversity and composition), soil heterogeneity and spatial covariates on the spatial structure of the AM fungal communities in a semiarid Mediterranean scrubland. Furthermore, we evaluated how the phylogenetic relatedness of both plants and AM fungi shapes these symbiotic relationships. METHODS: We characterized the composition and diversity of AM fungal and plant communities in a dry Mediterranean scrubland taxonomically and phylogenetically, using DNA metabarcoding and a spatially explicit sampling design at the plant neighbourhood scale. KEY RESULTS: The above- and below-ground plant community attributes, soil physicochemical properties and spatial variables explained unique fractions of AM fungal diversity and composition. Mainly, variations in plant composition affected the AM fungal composition and diversity. Our results also showed that particular AM fungal taxa tended to be associated with closely related plant species, suggesting the existence of a phylogenetic signal. Although soil texture, fertility and pH affected AM fungal community assembly, spatial factors had a greater influence on AM fungal community composition and diversity than soil physicochemical properties. CONCLUSIONS: Our results highlight that the more easily accessible above-ground vegetation is a reliable indicator of the linkages between plant roots and AM fungi. We also emphasize the importance of soil physicochemical properties in addition to below-ground plant information, while accounting for the phylogenetic relationships of both plants and fungi, because these factors improve our ability to predict the relationships between AM fungal and plant communities.
Assuntos
Micorrizas , Micorrizas/genética , Ecossistema , Filogenia , Solo/química , Simbiose , Raízes de Plantas , Plantas/microbiologia , Microbiologia do Solo , FungosRESUMO
Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Domínio Catalítico , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Simulação de Acoplamento Molecular , Fosfoenolpiruvato Carboxiquinase (GTP)/químicaRESUMO
The Mycobacterium tuberculosis (Mtb) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodeling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen, it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, saturation transfer difference NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the synthesis of trehalose analogs. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substrate-specific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogs and specifies a framework that can be exploited for the design of new antitubercular agents and/or diagnostic tools.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Trealose/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Parede Celular/genética , Parede Celular/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Mutação , Mycobacterium tuberculosis/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Trealose/análogos & derivados , VirulênciaRESUMO
The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3'-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.
Assuntos
Proteínas de Bactérias/metabolismo , Clostridiales/metabolismo , Microbioma Gastrointestinal , alfa-L-Fucosidase/metabolismo , Proteínas de Bactérias/química , Clostridiales/química , Clostridiales/enzimologia , Trato Gastrointestinal/microbiologia , Glicoconjugados/metabolismo , Humanos , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Especificidade por Substrato , alfa-L-Fucosidase/químicaRESUMO
The human gut symbiont Ruminococcus gnavus scavenges host-derived N-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D NMR, we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro-N-acetylneuraminic acid intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram-negative and Gram-positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionization spray MS that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli depended on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.
Assuntos
Proteínas de Bactérias/química , Clostridiales/enzimologia , Ácido N-Acetilneuramínico/química , Oxirredutases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridiales/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Teste de Complementação Genética , Humanos , Mucinas/química , Mucinas/metabolismo , Ácido N-Acetilneuramínico/genética , Ácido N-Acetilneuramínico/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
ß-1â4-Glucan polysaccharides like cellulose, derivatives and analogues, are attracting attention due to their unique physicochemical properties, as ideal candidates for many different applications in biotechnology. Access to these polysaccharides with a high level of purity at scale is still challenging, and eco-friendly alternatives by using enzymes inâ vitro are highly desirable. One prominent candidate enzyme is cellodextrin phosphorylase (CDP) from Ruminiclostridium thermocellum, which is able to yield cellulose oligomers from short cellodextrins and α-d-glucose 1-phosphate (Glc-1-P) as substrates. Remarkably, its broad specificity towards donors and acceptors allows the generation of highly diverse cellulose-based structures to produce novel materials. However, to fully exploit this CDP broad specificity, a detailed understanding of the molecular recognition of substrates by this enzyme in solution is needed. Herein, we provide a detailed investigation of the molecular recognition of ligands by CDP in solution by saturation transfer difference (STD) NMR spectroscopy, tr-NOESY and protein-ligand docking. Our results, discussed in the context of previous reaction kinetics data in the literature, allow a better understanding of the structural basis of the broad binding specificity of this biotechnologically relevant enzyme.
Assuntos
Clostridium thermocellum , Glucosiltransferases , Espectroscopia de Ressonância Magnética , PolissacarídeosRESUMO
Understanding the fine details of the self-assembly of building blocks into complex hierarchical structures represents a major challenge en route to the design and preparation of soft-matter materials with specific properties. Enzymatically synthesised cellodextrins are known to have limited water solubility beyond DP9, a point at which they self-assemble into particles resembling the antiparallel celluloseâ II crystalline packing. We have prepared and characterised a series of site-selectively fluorinated cellodextrins with different degrees of fluorination and substitution patterns by chemoenzymatic synthesis. Bearing in mind the potential disruption of the hydrogen-bond network of celluloseâ II, we have prepared and characterised a multiply 6-fluorinated cellodextrin. In addition, a series of single site-selectively fluorinated cellodextrins was synthesised to assess the structural impact upon the addition of one fluorine atom per chain. The structural characterisation of these materials at different length scales, combining advanced NMR spectroscopy and microscopy methods, showed that a 6-fluorinated donor substrate yielded multiply 6-fluorinated cellodextrin chains that assembled into particles presenting morphological and crystallinity features, and intermolecular interactions, that are unprecedented for cellulose-like materials.
RESUMO
Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique ß-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host-microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.
Assuntos
Proteínas de Bactérias/química , Microbioma Gastrointestinal , Limosilactobacillus reuteri/fisiologia , Interações Microbianas , Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Animais , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Células Epiteliais/microbiologia , Concentração de Íons de Hidrogênio , Limosilactobacillus reuteri/química , Camundongos , Simulação de Dinâmica Molecular , Pectinas/metabolismo , Dobramento de Proteína , Sequências Repetitivas de Aminoácidos , Homologia de Sequência de Aminoácidos , SerinaRESUMO
Roots are assumed to play a major role in structuring soil microbial communities, but most studies exploring the relationships between microbes and plants at the community level have only used aboveground plant distribution as a proxy. However, a decoupling between belowground and aboveground plant components may occur due to differential spreading of plant canopies and root systems. Thus, soil microbe-plant links are not completely understood. Using a combination of DNA metabarcoding and spatially explicit sampling at the plant neighbourhood scale, we assessed the influence of the plant root community on soil bacterial and fungal diversity (species richness, composition and ß-diversity) in a dry Mediterranean scrubland. We found that root composition and biomass, but not richness, predict unique fractions of variation in microbial richness and composition. Moreover, bacterial ß-diversity was related to root ß-diversity, while fungal ß-diversity was related to aboveground plant ß-diversity, suggesting that plants differently influence both microbial groups. Our study highlights the role of plant distribution both belowground and aboveground, soil properties and other spatially structured factors in explaining the heterogeneity in soil microbial diversity. These results also show that incorporating data on both plant community compartments will further our understanding of the relationships between soil microbial and plant communities.
Assuntos
Biodiversidade , Solo , Bactérias/genética , Fungos , Raízes de Plantas , Microbiologia do SoloRESUMO
Ligand-based NMR techniques to study protein-ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi-frequency STD NMR approach, differential epitope mapping (DEEP)-STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so-called DEEP-STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunitâ B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP-STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi-subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.
Assuntos
Toxina da Cólera/química , Toxina da Cólera/metabolismo , Gangliosídeo G(M1)/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Sítios de Ligação , Ligantes , Ligação ProteicaRESUMO
Surface hydrophobization of cellulose nanomaterials has been used in the development of nanofiller-reinforced polymer composites and formulations based on Pickering emulsions. Despite the well-known effect of hydrophobic domains on self-assembly or association of water-soluble polymer amphiphiles, very few studies have addressed the behavior of hydrophobized cellulose nanomaterials in aqueous media. In this study, we investigate the properties of hydrophobized cellulose nanocrystals (CNCs) and their self-assembly and amphiphilic properties in suspensions and gels. CNCs of different hydrophobicity were synthesized from sulfated CNCs by coupling primary alkylamines of different alkyl chain lengths (6, 8, and 12 carbon atoms). The synthetic route permitted the retention of surface charge, ensuring good colloidal stability of hydrophobized CNCs in aqueous suspensions. We compare surface properties (surface charge, ζ potential), hydrophobicity (water contact angle, microenvironment probing using pyrene fluorescence emission), and surface activity (tensiometry) of different hydrophobized CNCs and hydrophilic CNCs. Association of hydrophobized CNCs driven by hydrophobic effects is confirmed by X-ray scattering (SAXS) and autofluorescent spectroscopy experiments. As a result of CNC association, CNC suspensions/gels can be produced with a wide range of rheological properties depending on the hydrophobic/hydrophilic balance. In particular, sol-gel transitions for hydrophobized CNCs occur at lower concentrations than hydrophilic CNCs, and more robust gels are formed by hydrophobized CNCs. Our work illustrates that amphiphilic CNCs can complement associative polymers as modifiers of rheological properties of water-based systems.
Assuntos
Celulose , Nanopartículas , Géis , Espalhamento a Baixo Ângulo , Suspensões , Água , Difração de Raios XRESUMO
We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by â¼twofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP- or ATP+ ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain.
Assuntos
Ginsenosídeos/farmacologia , Receptores Purinérgicos P2X4/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Benzoxazóis/metabolismo , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Células HEK293 , Humanos , Ivermectina/farmacologia , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Compostos de Quinolínio/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Sapogeninas/farmacologiaRESUMO
Lactobacillus reuteri is a gut symbiont inhabiting the gastrointestinal tract of numerous vertebrates. The surface-exposed serine-rich repeat protein (SRRP) is a major adhesin in Gram-positive bacteria. Using lectin and sugar nucleotide profiling of wild-type or L. reuteri isogenic mutants, MALDI-ToF-MS, LC-MS and GC-MS analyses of SRRPs, we showed that L. reuteri strains 100-23C (from rodent) and ATCC 53608 (from pig) can perform protein O-glycosylation and modify SRRP100-23 and SRRP53608 with Hex-Glc-GlcNAc and di-GlcNAc moieties, respectively. Furthermore, in vivo glycoengineering in E. coli led to glycosylation of SRRP53608 variants with α-GlcNAc and GlcNAcß(1â6)GlcNAcα moieties. The glycosyltransferases involved in the modification of these adhesins were identified within the SecA2/Y2 accessory secretion system and their sugar nucleotide preference determined by saturation transfer difference NMR spectroscopy and differential scanning fluorimetry. Together, these findings provide novel insights into the cellular O-protein glycosylation pathways of gut commensal bacteria and potential routes for glycoengineering applications.
Assuntos
Adesinas Bacterianas/química , Limosilactobacillus reuteri/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Glicosilação , Limosilactobacillus reuteri/genética , Limosilactobacillus reuteri/metabolismo , Mutação , Ressonância Magnética Nuclear Biomolecular , Sequências Repetitivas de AminoácidosRESUMO
A method for the direct measurement of free Ca2+ and Mg2+ concentrations in the range of 1-100 mM by NMR spectroscopy is demonstrated. The method automatically corrects for the effect of ionic strength on the activity of the species in solution and works satisfactorily even when significant concentrations of competitive ions are present. The method requires only the measurement of the 1H chemical shifts of our reporter ligands, glycolate and sulfoacetate, and is easily implemented using NMR imaging techniques. As a proof of concept, we extract the thermodynamic binding constants and conformer distributions of analyte ligands using an in situ ion gradient. Existing approaches for the measurement of free Ca2+ or Mg2+ concentrations by NMR operate only at very low ion concentrations or else require careful recalibration for different sample conditions. By providing the free Ca2+ or Mg2+ concentrations, the proposed methodology significantly enhances the information obtainable via NMR investigations of ion-responsive systems.
RESUMO
Differential epitope mapping saturation transfer difference (DEEP-STD) NMR spectroscopy is a recently developed powerful approach for elucidating the structure and pharmacophore of weak protein-ligand interactions, as it reports key information on the orientation of the ligand and the architecture of the binding pocket. The method relies on selective saturation of protein residues in the binding site and the generation of a differential epitope map by observing the ligand, which depicts the nature of the protein residues making contact with the ligand in the bound state. Selective saturation requires knowledge of the chemical-shift assignment of the protein residues, which can be obtained either experimentally by NMR spectroscopy or predicted from 3D structures. Herein, we propose a simple experimental procedure to expand the DEEP-STD NMR methodology to protein-ligand cases in which the spectral assignment of the protein is not available. This is achieved by experimentally identifying the chemical shifts of the residues present in binding hot-spots on the surface of the receptor protein by using 2D NMR experiments combined with a paramagnetic probe.