RESUMO
We advance the quality of first-principles calculations of protein electronic circular dichroism (CD) through an amelioration of a key deficiency of a previous procedure that involved diabatization of electronic states on the amide chromophore (to obtain interamide couplings) in a ß-strand conformation of a diamide. This yields substantially improved calculated far-ultraviolet (far-UV) electronic circular dichroism (CD) spectra for ß-sheet conformations. The interamide couplings from the diabatization procedure for 13 secondary structural elements (13 diamide structures) are applied to compute the CD spectra for seven example proteins: myoglobin (α helix), jacalin (ß strand), concanavalin A (ß type I), elastase (ß type II), papain (α + ß), 310-helix bundle (310-helix) and snow flea antifreeze protein (polyproline). In all cases, except concanavalin A and papain, the CD spectra computed using the interamide couplings from the diabatization procedure yield improved agreement with experiment with respect to previous first-principles calculations.
Assuntos
Dicroísmo Circular , Concanavalina A , Concanavalina A/química , Mioglobina/química , Proteínas/química , Papaína/química , Papaína/metabolismo , Peptídeos/química , Elétrons , Elastase Pancreática/química , Elastase Pancreática/metabolismo , Estrutura Secundária de ProteínaRESUMO
Leptospirosis is a global public health problem caused by Gram-negative pathogenic bacteria belonging to the genus Leptospira. The disease is transmitted through the urine of infected animals, which contaminates water and soil, leading to the infection of other animals and humans. Currently, several approaches exist to detect these bacteria; however, a new sensitive method for the live-cell imaging of Leptospira is required. In this study, we report the green synthesis of cadmium telluride quantum dots (CdTe QDs) which are unique fluorescent nanocrystals with a high fluorescence quantum yield capable of modifying cell surfaces and are biocompatible with cells. The fabrication of QDs with concanavalin A (ConA), a carbohydrate-binding lectin and known biological probe for Gram-negative bacteria, produced ConA-QDs which can effectively bind on Leptospira and exhibit strong fluorescence under simple fluorescence microscopy, allowing the live-cell imaging of the bacteria. Overall, we performed the simple synthesis of ConA-QDs and demonstrated their potential use as versatile fluorescent probes for the live-cell imaging of Leptospira. This technique could be further applied to track leptospiral cells and study the infection mechanism, contributing to a more thorough understanding of leptospirosis and how to control it in the future.
Assuntos
Leptospira , Pontos Quânticos , Pontos Quânticos/química , Corantes Fluorescentes/química , Compostos de Cádmio/química , Telúrio/química , Concanavalina A/química , Canavalia/química , Materiais Biocompatíveis/química , Microscopia de FluorescênciaRESUMO
Modular supramolecular complexes, where different proteins are assembled to gather targeting capability and photofunctional properties within the same structures, are of special interest for bacterial photodynamic inactivation, given their inherent biocompatibility and flexibility. We have recently proposed one such structure, exploiting the tetrameric bacterial protein streptavidin as the main building block, to target S. aureus protein A. To expand the palette of targets, we have linked biotinylated Concanavalin A, a sugar-binding protein, to a methylene blue-labelled streptavidin. By applying a combination of spectroscopy and microscopy, we demonstrate the binding of Concanavalin A to the walls of Gram-positive S. aureus and Gram-negative E. coli. Photoinactivation is observed for both bacterial strains in the low micromolar range, although the moderate affinity for the molecular targets and the low singlet oxygen yields limit the overall efficiency. Finally, we apply a maximum entropy method to the analysis of autocorrelation traces, which proves particularly useful when interpreting signals measured for diffusing systems heterogeneous in size, such as fluorescent species bound to bacteria.
Assuntos
Parede Celular , Concanavalina A , Escherichia coli , Staphylococcus aureus , Concanavalina A/química , Concanavalina A/metabolismo , Escherichia coli/metabolismo , Staphylococcus aureus/metabolismo , Parede Celular/metabolismo , Estreptavidina/química , Estreptavidina/metabolismo , Proteínas de Bactérias/metabolismo , Ligação ProteicaRESUMO
Infections caused by microorganisms are a public health problem worldwide. New biodetection systems are essential to diagnose with accuracy resulting in more effective treatment. In this work, we propose a ConA-conjugated graphene quantum dots and polypyrrole film-based biosensor for label-free detection of Candida albicans, Candida glabrata, Candida tropicalis, E. coli, B. subitilis, and S. aureus. We modified polypyrrole and graphene quantum dots (PPY-QDGs) with Concanavalin A (Con A) lectin. ConA is a glucose/mannose-specific lectin. The results showed that ConA lectin has the highest binding affinity for C. tropicalis and S. subtilis. PPY-GQDs-ConA binding profile revealed differential response for Candida spp (C. tropicalis > C. albicans > C. glabrata) and bacterial (B. subtilis > S. aureus > E. coli). The limits of detection (LOD) obtained were 1.42 CFU/mL for C. albicans, and 3.72 CFU/mL for C. glabrata. C. tropicalis yielded a LOD of 0.18 CFU/mL. The respective LODs for the evaluated bacteria were 0.39 CFU/mL for S. aureus, 0.72 CFU/mL for S. subtilis, and 2.63 CFU/mL for E. coli. The differential response obtained for the sensor can be attributed to the heterogeneous distribution of carbohydrates on the microorganism's surfaces. The proposed system based on a flexible substrate is effective for microbiological diagnosis.
Assuntos
Técnicas Biossensoriais , Concanavalina A , Grafite , Limite de Detecção , Polímeros , Pirróis , Pontos Quânticos , Grafite/química , Concanavalina A/química , Pontos Quânticos/química , Pirróis/química , Técnicas Biossensoriais/métodos , Polímeros/química , Candida/isolamento & purificação , Eletrodos , Técnicas Eletroquímicas/métodos , Escherichia coli/isolamento & purificação , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Kainate receptors, a subclass of ionotropic glutamate receptors, are tetrameric ligand-gated ion channels that mediate excitatory neurotransmission1-4. Kainate receptors modulate neuronal circuits and synaptic plasticity during the development and function of the central nervous system and are implicated in various neurological and psychiatric diseases, including epilepsy, depression, schizophrenia, anxiety and autism5-11. Although structures of kainate receptor domains and subunit assemblies are available12-18, the mechanism of kainate receptor gating remains poorly understood. Here we present cryo-electron microscopy structures of the kainate receptor GluK2 in the presence of the agonist glutamate and the positive allosteric modulators lectin concanavalin A and BPAM344. Concanavalin A and BPAM344 inhibit kainate receptor desensitization and prolong activation by acting as a spacer between the amino-terminal and ligand-binding domains and a stabilizer of the ligand-binding domain dimer interface, respectively. Channel opening involves the kinking of all four pore-forming M3 helices. Our structures reveal the molecular basis of kainate receptor gating, which could guide the development of drugs for treatment of neurological disorders.
Assuntos
Concanavalina A , Microscopia Crioeletrônica , Receptor de GluK2 Cainato , Ácido Glutâmico , Ativação do Canal Iônico , Modelos Moleculares , Domínios Proteicos , Receptores de Ácido Caínico , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/metabolismo , Receptores de Ácido Caínico/ultraestrutura , Humanos , Ácido Glutâmico/metabolismo , Ácido Glutâmico/química , Animais , Concanavalina A/química , Concanavalina A/metabolismo , Concanavalina A/farmacologia , Ligantes , Regulação Alostérica , Sítios de LigaçãoRESUMO
Macroporous cryogels are attractive scaffolds for biomedical applications, such as biomolecular immobilization, diagnostic sensing, and tissue engineering. In this study, thiol-reactive redox-responsive cryogels with a porous structure are prepared using photopolymerization of a pyridyl disulfide poly(ethylene glycol) methacrylate (PDS-PEG-MA) monomer. Reactive cryogels are produced using PDS-PEG-MA and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers, along with a PEG-based cross-linker and photoinitiator. Functionalization of cryogels using a fluorescent dye via the disulfide-thiol exchange reactions is demonstrated, followed by release under reducing conditions. For ligand-mediated protein immobilization, first, thiol-containing biotin or mannose is conjugated onto the cryogels. Subsequently, fluorescent dye-labeled proteins streptavidin and concanavalin A (ConA) are immobilized via ligand-mediated conjugation. Furthermore, we demonstrate that the mannose-decorated cryogel could capture ConA selectively from a mixture of lectins. The efficiency of protein immobilization could be easily tuned by changing the ratio of the thiol-sensitive moiety in the scaffold. Finally, an integrin-binding cell adhesive peptide is attached to cryogels to achieve successful attachment, and the on-demand detachment of integrin-receptor-rich fibroblast cells is demonstrated. Redox-responsive cryogels can serve as potential scaffolds for a variety of biomedical applications because of their facile synthesis and modification.
Assuntos
Criogéis , Oxirredução , Polietilenoglicóis , Criogéis/química , Polietilenoglicóis/química , Animais , Concanavalina A/química , Concanavalina A/metabolismo , Metacrilatos/química , Camundongos , Manose/química , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Compostos de Sulfidrila/química , Estreptavidina/química , Estreptavidina/metabolismo , Proteínas/química , Proteínas/metabolismo , Biotina/química , Biotina/metabolismo , Biotina/análogos & derivados , PorosidadeRESUMO
Lectin Con A, with specificity to interact with α-d-mannopyranoside, achieves tight binding affinity with the aid of optimal multivalent ligand valencies, distances and orientations between the ligands. A series of synthetic arabinomannans, possessing arabinan core and mannan at the non-reducing ends, is studied to assess the above constraints involved with lectin binding in this report. Trisaccharides, with (1 â 2)(1 â 3), (1 â 2)(1 â 5) and (1 â 3)(1 â 5) glycosidic bond connectivities, and a pentasaccharide with mannopyranosides at the non-reducing ends are synthesized. The binding affinities of the mannose bivalent ligands are studied with tetrameric Con A lectin by isothermal titration calorimetry (ITC). Among the derivatives, trisaccharide with (1 â 2)(1 â 3) glycosidic bond connectivity and the pentasaccharide undergo lectin interaction, clearly fulfilling the bivalent structural and functional valencies. Remaining oligosaccharides exhibit only a functional monovalency, defying the bivalent structural valency. The trisaccharide fulfilling the structural and functional valencies represent the smallest bivalent ligand, undergoing the lectin interaction in a trans-mode.
Assuntos
Lectinas , Mananas , Lectinas/química , Ligantes , Concanavalina A/química , Manose/química , Glicosídeos/química , Oligossacarídeos , Trissacarídeos , Ligação ProteicaRESUMO
La concanavalina A es una lectina con múltiples aplicaciones en Medicina, Biología, Hematología, Inmunología y en Bioquímica. Esta lectina representa una valiosa herramienta para el estudio, aislammiento y purificación de diversas proteínas. La concanavalina A se purificó por un método de absorción específica en sephadex G-50, a partir de Canavalia ensiformis cosechada en Cuba. El rendimiento obtenido fue de 1,56 porciento. El producto mostró una actividad hemoaglutinadora de eritrocitos humanos menor que 3,91 µg, y una actividad turbidimétrica de 1,75 mg. Se realizaron estudios de enfoque isoeléctrico y electroforético al producto purificado, y por ambos métodos resultó ser idéntico al de la firma FLOW. La connavallina A se acopló a sefarosa 4B activada con bromuro de cianógeno, y la matriz de afinidad obtenida se utilizó en la purificación de una hormona glicoproteíca marcada radiactivamente con yodo-125 con resultados satisfactorios