RESUMEN
Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.
Asunto(s)
Antígenos CD4 , Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas , Humanos , Antígenos CD4/metabolismo , Antígenos CD4/química , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , VIH-1/metabolismo , VIH-1/química , Simulación del Acoplamiento Molecular , Estabilidad Proteica , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.
Asunto(s)
Evolución Biológica , Vías Biosintéticas , Supervivencia Celular , Mutación , ProlinaRESUMEN
Salivary pH serves as a valuable and useful diagnostic marker for periodontal disease, as it not only plays a critical role in disease prevention but also in its development. Typically, saliva sampling is collected by draining and spitting it into collection tubes or using swabs. In this study, we have developed a Point-of-Care (POC) device for in situ determination of oral pH without the need for complex instruments, relying solely on a smartphone as the detection device. Our system utilizes a non-toxic vegetable colourimetric indicator, immobilized on a chitosan membrane located on a disposable stick, enabling direct sampling within the buccal cavity. An ad hoc designed 3D-printed attachment is used to ensure accurate positioning and alignment of the stick, as well as isolation from external lighting conditions. A custom-developed smartphone application captures and automatically processes the image of the sensing membrane, providing the salivary pH results. After optimizing the cocktail composition, the developed sensors demonstrated the capacity to determine pH within a range of 5.4 to 8.1 with a remarkable precision of 0.6%, achieving a very short analysis time of just 1 min. A stability study conducted on the sensing membranes revealed a lifetime of 50 days. To validate the performance of our analytical device, we compared its results against those obtained from a calibrated pH-meter, using a group of individuals. The device exhibited an average error of 2.4% when compared with the pH-meter results, confirming its reliability and accuracy.
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Quitosano , Teléfono Inteligente , Humanos , Sistemas de Atención de Punto , Reproducibilidad de los Resultados , Concentración de Iones de HidrógenoRESUMEN
Trans-sialidases (TS) are important constitutive macromolecules of the secretome present on the surface of Trypanosoma cruzi (T. cruzi) that play a central role as a virulence factor in Chagas disease. These enzymes have been related to infectivity, escape from immune surveillance and pathogenesis exhibited by this protozoan parasite. In this work, atomic force microscopy (AFM)-based single molecule-force spectroscopy is implemented as a suitable technique for the detection and location of functional TS on the surface of extracellular vesicles (EVs) released by tissue-culture cell-derived trypomastigotes (Ex-TcT). For that purpose, AFM cantilevers with functionalized tips bearing the anti-TS monoclonal antibody mAb 39 as a sense biomolecule are engineered using a covalent chemical ligation based on vinyl sulfonate click chemistry; a reliable, simple and efficient methodology for the molecular recognition of TS using the antibody-antigen interaction. Measurements of the breakdown forces between anti-TS mAb 39 antibodies and EVs performed to elucidate adhesion and forces involved in the recognition events demonstrate that EVs isolated from tissue-culture cell-derived trypomastigotes of T. cruzi are enriched in TS. Additionally, a mapping of the TS binding sites with submicrometer-scale resolution is provided. This work represents the first AFM-based molecular recognition study of Ex-TcT using an antibody-tethered AFM probe.
Asunto(s)
Vesículas Extracelulares , Parásitos , Trypanosoma cruzi , Animales , Vesículas Extracelulares/metabolismo , Glicoproteínas , Microscopía de Fuerza Atómica , Neuraminidasa/metabolismo , Parásitos/metabolismo , Trypanosoma cruzi/metabolismoRESUMEN
A microfluidic paper-based analytical device integrating carbon dot (CDs) is fabricated and used for a fluorometric off-on assay of biothiols. Vinyl sulfone (VS) click immobilization of carbon dots (CDs) on paper was accomplished by a one-pot simplified protocol that uses divinyl sulfone (DVS) as a homobifunctional reagent. This reagent mediated both the click oxa-Michael addition to the hydroxyl groups of cellulose and ulterior covalent grafting of the resulting VS paper to NH2-functionalized CDs by means of click aza-Michael addition. The resulting cellulose nanocomposite was used to engineer an inexpensive and robust microfluidic paper-based analytical device (µPAD) that is used for a reaction-based off-on fluorometric assay of biothiols (GSH, Cys, and Hcy). The intrinsic blue fluorescence of CDs (with excitation/emission maxima at 365/450 nm) is turned off via the heavy atom effect of an introduced iodo group. Fluorescence is turned on again due to the displacement of iodine by reaction with a biothiol. The increase in fluorescence is related to the concentration over a wide range (1 to 200 µM for GSH and 5-200 µM for Cys and Hcy, respectively), and the assay exhibits a low detection limit (0.3 µM for GSH and Cys and 0.4 µM for Hcy). The method allows for rapid screening and can also be used in combination with a digital camera readout. Graphical abstract Schematic representation of a µPAD based on click immobilized carbon dots and used for a reaction-based fluorometric off-on assay of biothiols. The intrinsic blue fluorescence of carbon dots is turned off via the heavy atom effect of an introduced iodo group and turned on by the displacement of this atom by reaction with a biothiol.
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Cisteína/orina , Glutatión/orina , Homocisteína/orina , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , Puntos Cuánticos/química , Carbono/química , Química Clic , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Papel , Espectrometría de Fluorescencia/métodos , Sulfonas/químicaRESUMEN
Polymer-based nanotheranostics are appealing tools for cancer treatment and diagnosis in the fast-growing field of nanomedicine. A straightforward preparation of novel engineered PEI-based nanotheranostics incorporating NIR fluorescence heptamethine cyanine dyes (NIRF-HC) to enable them with tumor targeted gene delivery capabilities is reported. Branched PEI-2 kDa (b2kPEI) is conjugated with IR-780 and IR-783 dyes by both covalent and noncovalent simple preparative methodologies varying their stoichiometry ratio. The as-prepared set of PEI-NIR-HC nanocarriers are assayed in vitro and in vivo to evaluate their gene transfection efficiency, cellular uptake, cytotoxicity, internalization and trafficking mechanisms, subcellular distribution, and tumor specific gene delivery. The results show the validity of the approach particularly for one of the covalent IR783-b2kPEI conjugates that exhibit an enhanced tumor uptake, probably mediated by organic anion transporting peptides, and favorable intracellular transport to the nucleus. The compound behaves as an efficient nanotheranostic transfection agent in NSG mice bearing melanoma G361 xenographs with concomitant imaging signal and gene concentration in the targeted tumor. By this way, advanced nanotheranostics with multifunctional capabilities (gene delivery, tumor-specific targeting, and NIR fluorescence imaging) are generated in which the NIRF-HC dye component accounts for simultaneous targeting and diagnostics, avoiding additional incorporation of additional tumor-specific targeting bioligands.
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Carbocianinas/administración & dosificación , Nanomedicina , Polietileneimina/química , Espectroscopía Infrarroja Corta/métodos , Nanomedicina Teranóstica , Animales , Línea Celular , Fluorescencia , Técnicas de Transferencia de Gen , RatonesRESUMEN
Gene transfection mediated by the cationic polymer polyethylenimine (PEI) is considered a standard methodology. However, while highly branched PEIs form smaller polyplexes with DNA that exhibit high transfection efficiencies, they have significant cell toxicity. Conversely, low molecular weight PEIs (LMW-PEIs) with favorable cytotoxicity profiles display minimum transfection activities as a result of inadequate DNA complexation and protection. To solve this paradox, a novel polyelectrolyte complex was prepared by the ionic cross-linking of branched 1.8 kDa PEI with citric acid (CA). This system synergistically exploits the good cytotoxicity profile exhibited by LMW-PEI with the high transfection efficiencies shown by highly branched and high molecular weight PEIs. The polyectrolyte complex (1.8 kDa-PEI@CA) was obtained by a simple synthetic protocol based on the microwave irradiation of a solution of 1.8 kDa PEI and CA. Upon complexation with DNA, intrinsic properties of the resulting particles (size and surface charge) were measured and their ability to form stable polyplexes was determined. Compared with unmodified PEIs the new complexes behave as efficient gene vectors and showed enhanced DNA binding capability associated with facilitated intracellular DNA release and enhanced DNA protection from endonuclease degradation. In addition, while transfection values for LMW-PEIs are almost null, transfection efficiencies of the new reagent range from 2.5- to 3.8-fold to those of Lipofectamine 2000 and 25 kDa PEI in several cell lines in culture such as CHO-k1, FTO2B hepatomas, L6 myoblasts, or NRK cells, simultaneously showing a negligible toxicity. Furthermore, the 1.8 kDa-PEI@CA polyelectrolyte complexes retained the capability to transfect eukaryotic cells in the presence of serum and exhibited the capability to promote in vivo transfection in mouse (as an animal model) with an enhanced efficiency compared to 25 kDa PEI. Results support the polyelectrolyte complex of LMW-PEI and CA as promising generic nonviral gene carriers.
Asunto(s)
Ácido Cítrico/química , Electrólitos/química , Polietileneimina/química , Transfección , Línea Celular , Humanos , Técnicas In Vitro , Peso MolecularRESUMEN
Sutures are commonly used for surgical procedures and new sutures are being developed to improve wound healing. In the past decade, it has been extensively shown that mesenchymal stem cells (MSCs) have a wound healing potential. To benefit the overall wound healing process, we aimed to analyze the usage of pretreated sutures for improving the implantation of MSCs in the tissues. Our results firstly showed that suture pretreatments with gelatin, poly-L-lysine, and NaOH improved the adhesive strength of MSCs to sutures. These cells remained surrounding the sutured tissue and no significant phenotypic changes were found in those cells cultured onto pretreated sutures. In vivo experiments showed that the implantation of MSCs by suturing increases the collagen content in the sutured tissue. Moreover, proteomics analysis of secreted proteins showed that collagen alpha-1(I) chain was the most abundant collagen found. To our knowledge, this is the first report that aimed to improve the implantation of MSCs in tissue by suture pretreatments. Moreover, in vivo experiments suggest that MSC-coated sutures may enhance wound healing and tissue remodeling through the release of different collagen types being applicable for those patients that tend to have difficulty healing.
Asunto(s)
Colágeno/metabolismo , Células Madre Mesenquimatosas , Piel/patología , Suturas , Cicatrización de Heridas , Animales , Modelos Animales de Enfermedad , Citometría de Flujo , Gelatina/farmacología , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Polietilenglicoles/farmacología , Polilisina/análogos & derivados , Polilisina/farmacología , Piel/lesiones , Piel/metabolismo , Resistencia a la TracciónRESUMEN
Water recycling and reuse are cornerstones of water management, which can be compromised by the presence of pollutants. Among these, pharmaceuticals can overcome standard water treatments and require sophisticated approaches to remove them. Sorption is an economically viable alternative limited by the need for sorbents with a sorption coefficient (Kd) higher than 500 L/kg. The cross-linking of dextrin (Dx) with divinyl sulfone (DVS) in the presence of 1 mmol or 5 mmol of ibuprofen (IBU) yields the insoluble polymers pDx1 and pDx5 with improved affinity for IBU and high selectivity towards erythromycin (ERY) and ERY Kd higher than 4 × 103 L/kg, when tested against a cocktail of six drugs. Characterization of the polymers shows that both pDx1 and pDx5 have similar properties, fast sorption kinetics, and ERY Kd of 13.3 × 103 for pDx1 and 6.4 × 103 for pDx5, representing 26.6 and 12.0 times the 500 L/kg threshold. The fact that new affinities and improvements in Kd can be achieved by cross-linking Dx in the presence of other molecules that promote pre-organization expands the applications of DVS cross-linked polysaccharides as sustainable, scalable, and environmentally friendly sorbents with a potential application in wastewater treatment plants (WTPs).
RESUMEN
BACKGROUND: S-nitrosylaton is implicated in the regulation of numerous signaling pathways with a diversity of regulatory roles. The high lability of the S-NO bond makes the study of proteins regulated by S-nitrosylation/denitrosylation a challenging task and most studies have focused on already S-nitrosylated proteins. We hypothesize that: i) S-nitrosoglutathione (GSNO) transnitrosylation is a feasible mechanism to account for the physiological S-nitrosylation of rather electropositive sulfur atoms from proteins, ii) affinity chromatography is a suitable approach to isolate proteins that are prone to undergo S-transnitrosylation and iii) vinyl sulfone silica is a suitable chromatographic bead. RESULTS: The combination of vinyl sulfone silica with GSNO yielded an affinity resin that withstood high ionic strength without shrinking or deforming and that it was suitable to isolate potential GSNO transnitrosylation target candidates. Fractions eluted at 1500 mM NaCl resulted in a symmetrical peak for both, protein and S-nitrosothiols, supporting the idea of transnitrosylation by GSNO as a selective process that involves strong and specific interactions with the target protein. Proteomic analysis led to the identification of 22 physiological significant enzymes that differ with the tissue analyzed, being regulatory proteins the most abundant group in hypocotyls. The identification of chloroplastidic FBPase, proteasome, GTP-binding protein, heat shock Hsp70, syntaxin, catalase I, thioredoxin peroxidase and cytochrome P450 that have already been reported as S-nitrosylated by other techniques can be considered as internal positive controls that validate our experimental approach. An additional validation was provided by the prediction of the S-nitrosylation sites in 19 of the GSNO transnitrosylation target candidates. CONCLUSIONS: Vinyl sulfone silica is an open immobilization support that can be turned ad hoc and in a straightforward manner into an affinity resin. Its potential in omic sciences was successfully put to test in the context of the analysis of post-translational modification by S-nitrosylation with two different tissues: mature pea leaves and embryogenic sunflower hypocotyls. The identified proteins reveal an intriguing overlap among S-nitrosylation and both tyrosine nitration and thioredoxin regulation. Chloroplastidic FBPase is a paradigm of such overlap of post-translational modifications since it is reversible modified by thioredoxin and S-nitrosylation and irreversibly by tyrosine nitration. Our results suggest a complex interrelation among different modulation mechanisms mediated by NO-derived molecules.
Asunto(s)
Cromatografía de Afinidad/métodos , Helianthus/metabolismo , Pisum sativum/metabolismo , Proteínas de Plantas/metabolismo , S-Nitrosoglutatión/metabolismo , Secuencias de Aminoácidos , Cromatografía de Afinidad/instrumentación , Helianthus/química , Pisum sativum/química , Proteínas de Plantas/química , Procesamiento Proteico-Postraduccional , Dióxido de Silicio/química , Sulfonas/químicaRESUMEN
Water use has been increasing globally by 1% per year, and recycling and re-use are critical issues compromised by the presence of pollutants. In this context, the design of novel materials and/or procedures for the large scale-removal of pollutants must be economically and environmentally feasible in order to be considered as part of the solution by emerging economies. We demonstrate that the cross-linking of biodegradable polysaccharides such as starch, dextrin, or dextrin and ß-cyclodextrin with divinyl sulfone is an innovative strategy for synthesizing insoluble and eco-friendly sorbent polymers, including pSt, pDx and pCD-Dx. The evaluation of these polymers' ability to remove ciprofloxacin (CIP), a prime example of antibiotic pollution, revealed that pSt, with a Kd of 1469 L/kg and a removal rate higher than 92%, is a favorable material. Its sorption is pH-dependent and enhanced at a mildly alkaline pH, allowing for the desorption (i.e., cleaning) and reuse of pSt through an environmentally friendly treatment with 20 mM AcONa pH 4.6. The facts that pSt (i) shows a high affinity for CIP even at high NaCl concentrations, (ii) can be obtained from affordable starting materials, and (iii) is synthesized and regenerated through organic, solvent-free procedures make pSt a novel sustainable material for inland water and seawater remediation, especially in less developed countries, due to its simplicity and low cost.
RESUMEN
Carbohydrate-mediated molecular recognition is involved in many biological aspects such as cellular adhesion, immune response, blood coagulation, inflammation, and infection. Considering the crucial importance of such biological events in which proteins are normally involved, synthetic saccharide-based systems have emerged as powerful tools for the understanding of protein-carbohydrate interactions. As a new approach to create saccharide-based systems, a set of representative monosaccharides (D-mannose, D-glucose, N-acetyl-D-glucosamine, and L-fucose) and disaccharides (lactose, maltose, and melibiose) were derivatized at their anomeric carbon with a vinyl sulfone group spanned by an ethylthio linker. This vinyl sulfone functionalization is demonstrated to be a general strategy for the covalent linkage of a saccharide in mild conditions via Michael-type additions with the amine and thiol groups from functionalized supports and those naturally present in biomolecules. The introduction of the ethylthio linker between the biorecognizable element (i.e., saccharide) and the reactive group (i.e., vinyl sulfone) was found to preserve the functionality of the former. The capability of the vinyl sulfone saccharides for the study of lectin-carbohydrate interactions was demonstrated by (i) immobilizing them on both amine-functionalized supports (glass slides and microwell plates) and polylysine-coated glass slides to create sugar arrays that selectively bind lectins (ii) coupling to model proteins to yield neoglycoproteins that are recognized by lectins and (iii) using vinyl sulfone saccharides as tags to allow the detection of the labeled biomolecule by HRP-lectins. The above results were further put tothe test with a real case: detection of carbohydrate binding proteins present in rice ( Oryza sativa ).
Asunto(s)
Metabolismo de los Hidratos de Carbono , Carbohidratos/química , Lectinas/metabolismo , Sulfonas/química , Animales , Técnicas Biosensibles , Bovinos , Estudios de Factibilidad , Vidrio/química , Glicoproteínas/síntesis química , Glicoproteínas/metabolismo , Unión ProteicaRESUMEN
A new approach for the preparation of carbohydrate-coated magnetic nanoparticles is reported. In a first step, we show that the pH-driven assembly-disassembly natural process that occurs in apoferritin protein is effective for the encapsulation of maghemite nanoparticles of different sizes: 4 and 6 nm. In a second step, we demonstrate that the presence of functional amine groups in the outer shell of apoferritin allows functionalization with two carbohydrates, N-acetyl-D-glucosamine and d-mannose. High-resolution electron microscopy (HREM), high angle annular dark field scanning electron microscopy (HAADF-STEM), electron energy loss spectroscopy (EELS), X-ray diffraction (XRD), and SQUID technique have been used to characterize the magnetic samples, termed herein Apomaghemites. The in vivo magnetic resonance imaging (MRI) studies showed the efficiency in contrasting images for these samples; that is, the r(2) NMR relaxivities are comparable with Endorem (a commercial superparamagnetic MRI contrast agent). The r(2) relaxivity values as well as the pre-contrast and post-contrast T(2)*-weighted images suggested that our systems could be used as perspective superparamagnetic contrast agents for magnetic resonance imaging (MRI). The carbohydrate-functionalized Apomaghemite nanoparticles retained their recognition abilities, as demonstrated by the strong affinity with their corresponding carbohydrate-binding lectins.
Asunto(s)
Apoferritinas/química , Compuestos Férricos/química , Lectinas/química , Magnetismo , Nanopartículas/química , Subunidades de Proteína/química , Carbohidratos/química , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Magnetite nanoparticles (MNPs) coated by branched poly (ethylene-imine) (PEI) were synthesized in a one-pot. Three molecular weights of PEI were tested, namely, 1.8 kDa (sample MNP-1), 10 kDa (sample MNP-2), and 25 kDa (sample MNP-3). The MNP-1 particles were further functionalized with folic acid (FA) (sample MNP-4). The four types of particles were found to behave magnetically as superparamagnetic, with MNP-1 showing the highest magnetization saturation. The particles were evaluated as possible hyperthermia agents by subjecting them to magnetic fields of 12 kA/m strength and frequencies ranging between 115 and 175 kHz. MNP-1 released the maximum heating power, reaching 330 W/g at the highest frequency, in the high side of reported values for spherical MNPs. In vitro cell viability assays of MNP-1 and MNP-4 against three cell lines expressing different levels of FA receptors (FR), namely, HEK (low expression), and HeLa (high expression), and HepG2 (high expression), demonstrated that they are not cytotoxic. When the cells were incubated in the presence of a 175 kHz magnetic field, a significant reduction in cell viability and clone formation was obtained for the high expressing FR cells incubated with MNP-4, suggesting that MNP-4 particles are good candidates for magnetic field hyperthermia and active targeting.
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The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due to their smaller size, low immunogenicity, and low-cost production. Although covalent strategies for the preparation of such ScFv-based therapeutic conjugates are prevalent, this approach is not straightforward, as it requires prior chemical activation and/or modification of both the ScFv and the therapeutics for the application of robust chemistries. A non-covalent alternative based on ScFv fused to maltose-binding protein (MBP) acting as a binding adapter is proposed for active targeted delivery. MBP-ScFv proves to be a valuable modular platform to synergistically bind maltose-derivatized therapeutic cargos through the MBP, while preserving the targeting competences provided by the ScFv. The methodology has been tested by using a mutated maltose-binding protein (MBP I334W) with an enhanced affinity toward maltose and an ScFv coding sequence toward the human epidermal growth factor receptor 2 (HER2). Non-covalent binding complexes of the resulting MBP-ScFv fusion protein with diverse maltosylated therapeutic cargos (a near-infrared dye, a maltosylated supramolecular ß-cyclodextrin container for doxorubicin, and non-viral polyplex gene vector) were easily prepared and characterized. In vitro and in vivo assays using cell lines that express or not the HER2 epitope, and mice xenografts of HER2 expressing cells demonstrated the capability and versatility of MBP-ScFv for diagnosis, imaging, and drug and plasmid active targeted tumor delivery. Remarkably, the modularity of the MBP-ScFv platform allows the flexible interchange of both the cargos and the coding sequence for the ScFv, allowing ad hoc solutions in targeting delivery without any further optimization since the MBP acts as a pivotal element.
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Anticuerpos de Cadena Única , Animales , Anticuerpos Monoclonales , Doxorrubicina , Maltosa , Proteínas de Unión a Maltosa/genética , Ratones , Anticuerpos de Cadena Única/genéticaRESUMEN
Multimeric lactosides based on carbohydrate scaffolds with valencies ranging from 1 to 4 and different linker lengths were synthesized by a copper-catalyzed azide-alkyne cycloaddition (CuAAC). The binding affinities and crosslinking abilities of the new "click clusters" toward biologically relevant galectins (gal-1, gal-3) and peanut lectin were evaluated by fluorescent polarization assay (FPA) and enzyme-linked lectin assay (ELLA), respectively. FPA indicated that the binding affinities of the synthetic multilactosides towards the galectins increased proportionally with their lactosyl content, without significant differences due to the spacer length. ELLA evidenced a modest cluster effect for the multivalent conjugates, with a relative potency per lactoside ranging from 2.1 to 3.2. Nearly identical binding affinities were recorded for derivatives differing in the length of the linkers, in agreement with the FPA data. These results demonstrate that this parameter does not significantly influence the recognition process when interactions occur at a single lectin site. Molecular dynamics revealed that glycoconjugates adopt a pseudoglobular structure with a random localization of the lactoside residues. These spatial distributions were observed irrespective of the linker length; this explains the virtually equal affinities recorded by ELLA. In contrast, two-site "sandwich" ELLA clearly revealed that multivalent derivatives bearing the longest spacers were more efficient for crosslinking lectins. Intrinsic affinities, devoid of aggregation effects, and crosslinking capabilities are, therefore, not directly related phenomena that must be taking into consideration in neoglycoconjugate design for specific applications.
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Galectina 1/química , Galectina 3/química , Glicósidos/química , Aglutinina de Mani/química , Alquinos/química , Arachis/metabolismo , Azidas/química , Catálisis , Cobre/química , Pruebas de Enzimas , Inmunoensayo de Polarización Fluorescente , Simulación de Dinámica MolecularRESUMEN
The easy functionalization of tags and solid supports with the vinyl sulfone function is a valuable tool in omic sciences that allows their coupling with the amine and thiol groups present in the proteogenic residues of proteins, in mild and green conditions compatible with their biological function.
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Proteínas Inmovilizadas/química , Sulfonas/química , Aminas/química , Animales , Bovinos , Concentración de Iones de Hidrógeno , Indicadores y Reactivos/química , Modelos Moleculares , Conformación Proteica , Proteínas/química , Rodaminas/química , Dióxido de Silicio/química , Coloración y Etiquetado , Sulfonas/síntesis química , TemperaturaRESUMEN
Chromophore-appended cyclodextrins combine the supramolecular loading capabilities of cyclodextrins (CDs) with the optical properties of the affixed chromophores. Among fluorescent materials, carbon dots (CNDs) are attractive and the feasibility of CND-appended CDs as sensors has been demonstrated by different authors. However, CNDs are intrinsically heterogeneous materials and their ulterior functionalization yields hybrid composites that are not well defined in terms of structure and composition. Inspired by the fluorescence properties of 5-oxo-1,2,3,5-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid (IPCA), the most paradigmatic of the molecular fluorophores detected in CNDs, herein we report two highly efficient synthetic chemical strategies for the preparation of IPCA-appended CDs that behave as CND-based CD "turn off-on" biosensors suitable for the analysis of cholesterol and ß-galactosidase activity. We have deconstructed the CND-CD systems to demonstrate that (i) the role of CNDs is limited to acting as a support for the molecular fluorophores produced during their synthesis and (ii) the molecular fluorophores suffice for the determination of the enzymatic activity based on the quenching by p-nitrophenol as a sacrificial quencher.
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Técnicas Biosensibles , Carbono/química , Ciclodextrinas/química , Puntos Cuánticos , Colesterol/sangre , Fluorescencia , Colorantes Fluorescentes/química , Humanos , Nitrofenoles/química , beta-Galactosidasa/análisisRESUMEN
Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains sluggish, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib is a novel approach which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of predicted stability. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this system particularly challenging to optimize. Yet, experimental screening of a small number of active-site variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers (â¼2 × 104 M-1 s-1 and â¼102 s-1) for this anthropogenic reaction that compare favorably to those of modern natural enzymes. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, even on scaffolds that were not originally evolved for those functions, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within â¼2 kcal mol-1 and indicate that the enhanced activity is linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stability-guidance of FuncLib by computational predictions of catalytic activity, as a generalized approach for computational enzyme design.
RESUMEN
Activated carbon nanodots functionalized with acid anhydride groups (AA-CNDs) are prepared by one-pot water-free green thermolysis of citric acid. As a proof of concept of their capabilities as appealing and versatile platforms for accessing engineering nanoconstructs, the as-prepared AA-CNDs have been reacted to yield clickable CNDs. Their click bioconjugation with relevant recognizable complementary clickable sugars has led to multivalent CND-based glyconanoparticles that are non-toxic and biorecognizable. The accessibility and intrinsic reactivity of AA-CNDs expand the current toolbox of covalent surface grafting methodologies and provide a wide range of potential applications for engineering (bio)nanoconstructs.