RESUMO
We introduce a novel and straightforward methodology for photoredox arylation of an indole scaffold using aryldiazonium salts under mild and metal-free conditions. Our approach enables the regioselective and chemoselective introduction of several aryl groups to the C(2) position of indoles and tryptophan, even in competition with other amino acids. This approach extends to the late-stage functionalization of peptides and lysozyme, heralding the unprecedented arylation of tryptophan residues in wild-type proteins and offering broad utility in chemical biology.
Assuntos
Indóis , Oxirredução , Triptofano , Triptofano/química , Indóis/química , Estrutura Molecular , Processos Fotoquímicos , Muramidase/química , Peptídeos/química , Estereoisomerismo , CatáliseRESUMO
The use of computer simulation for binding affinity prediction is growing in drug discovery. However, its wider use is constrained by the accuracy of the free energy calculations. The key sources of error are the force fields used to depict molecular interactions and insufficient sampling of the configurational space. To improve the quality of the force field, we developed a Python-based computational workflow. The workflow described here uses the minimal basis iterative stockholder (MBIS) method to determine atomic charges and Lennard-Jones parameters from the polarized molecular density. This is done by performing electronic structure calculations on various configurations of the ligand when it is both bound and unbound. In addition, we validated a simulation procedure that accounts for the protein and ligand degrees of freedom to precisely calculate binding free energies. This was achieved by comparing the self-adjusted mixture sampling and nonequilibrium thermodynamic integration methods using various protein and ligand conformations. The accuracy of predicting binding affinity is improved by using MBIS-derived force field parameters and a validated simulation procedure. This improvement surpasses the chemical precision for the eight aromatic ligands, reaching a root-mean-square error of 0.7 kcal/mol.
Assuntos
Muramidase , Ligação Proteica , Termodinâmica , Muramidase/química , Muramidase/metabolismo , Ligantes , Elétrons , Bacteriófago T4/enzimologia , Mutação , Conformação Proteica , Simulação de Dinâmica Molecular , Modelos MolecularesRESUMO
Amyloid aggregates arise from either the partial or complete loss of the native protein structure or the inability of proteins to attain their native conformation. These aggregates have been linked to several diseases, including Alzheimer's, Parkinson's, and lysozyme amyloidosis. A comprehensive dataset was recently reported, demonstrating the critical role of the protein's surrounding environment in amyloid formation. In this study, we investigated the formation of lysozyme amyloid fibrils induced by sodium dodecyl sulfate (SDS) and the effect of solvents in the medium. Experimental data obtained through fluorescence spectroscopy revealed a notable lag phase in amyloid formation when acetone solution was present. This finding suggested that the presence of acetone in the reaction medium created an unfavorable microenvironment for amyloid fibril formation and impeded the organization of the denatured protein into the fibril form. The in silico data provided insights into the molecular mechanism of the interaction between acetone molecules and the lysozyme protofibril, once acetone presented the best experimental results. It was observed that the lysozyme protofibril became highly unstable in the presence of acetone, leading to the complete loss of its ß-sheet conformation and resulting in an open structure. Furthermore, the solvation layer of the protofibril in acetone solution was significantly reduced compared to that in other solvents, resulting in fewer hydrogen bonds. Consequently, the presence of acetone facilitated the exposure of the hydrophobic portion of the protofibril, precluding the amyloid fibril formation. In summary, our study underscores the pivotal role the surrounding environment plays in influencing amyloid formation.
Assuntos
Amiloide , Muramidase , Dodecilsulfato de Sódio/química , Amiloide/química , Muramidase/química , Solventes/química , AcetonaRESUMO
PEGylated protein purification with the required quality attributes has represented a bioengineering challenge and Affinity Monolith Chromatography (AMC) has never been exploited for this goal. This work reports the generation of a heparin-modified affinity monolith disk by reductive alkylation with raised ligand density for its use as chromatographic support in the separation of lysozyme PEGylation reactions (LPRs) with three different PEG sizes (1, 20 and 40 kDa). For immobilized heparin determination a modified toluidine colorimetric assay adapted to microplate format was proposed. The heparin modified-disk was able to differentiate positional isomers of 20 kDa mono-PEGylated lysozyme at neutral pH using a salt linear gradient. Identity of PEG-conjugates was verified by SDS-PAGE and positional isomers were partially characterized by peptide mapping mass spectrometry. 20 kDa mono-PEGylated lysozyme conjugate purity (99.69 ± 0.05%) was comparable with traditional chromatographic methods while productivity (0.0964 ± 0.0001 mg/mL*min) was increased up to 6.1 times compared to that obtained in heparin packed-bed affinity chromatography procedures. The proposed AMC method represents a reliable, efficient, easy-handling, fast and single-step operation for the analysis or preparative isolation of PEGylated proteins containing a heparin binding domain.
Assuntos
Heparina , Muramidase , Antivirais , Cromatografia , Eletroforese em Gel de Poliacrilamida , Muramidase/química , Polietilenoglicóis/químicaRESUMO
This report describes the application of dielectric spectroscopy as a simple and fast way to guide protein adsorption experiments. Specifically, the polarization behavior of a layer of adsorbed lysozyme was investigated using a triangular-wave signal with frequencies varying from 0.5 to 2â Hz. The basic experiment, which can be performed in less than 5â min and with a single sample, not only allowed confirming the susceptibility of the selected protein towards the electric signal but also identified that this protein would respond more efficiently to signals with lower frequencies. To verify the validity of these observations, the adsorption behavior of lysozyme onto optically transparent carbon electrodes was also investigated under the influence of an applied alternating potential. In these experiments, the applied signal was defined by a sinusoidal wave with an amplitude of 100â mV and superimposed to +800â mV (applied as a working potential) and varying the frequency in the 0.1-10000â Hz range. The experimental data showed that the greatest adsorbed amounts of lysozyme were obtained at the lowest tested frequencies (0.1-1.0â Hz), results that are in line with the corresponding dielectric features of the protein.
Assuntos
Espectroscopia Dielétrica , Muramidase , Adsorção , Eletricidade , Eletrodos , Muramidase/químicaRESUMO
Glycation process refers to reactions between reduction sugars and amino acids that can lead to formation of advanced glycation end products (AGEs) which are related to changes in chemical and functional properties of biological structures that accumulate during aging and diseases. The aim of this study was to perform and analyze in vitro glycation by fructose and methylglyoxal (MGO) using salivary fluid, albumin, lysozyme, and salivary α-amylase (sAA). Glycation effect was analyzed by biochemical and spectroscopic methods. The results were obtained by fluorescence analysis, infrared spectroscopy (total attenuated reflection-Fourier transform, ATR-FTIR) followed by multivariate analysis of principal components (PCA), protein profile, immunodetection, enzymatic activity and oxidative damage to proteins. Fluorescence increased in all glycated samples, except in saliva with fructose. The ATR-FTIR spectra and PCA analysis showed structural changes related to the vibrational mode of glycation of albumin, lysozyme, and salivary proteins. Glycation increased the relative molecular mass (Mr) in protein profile of albumin and lysozyme. Saliva showed a decrease in band intensity when glycated. The analysis of sAA immunoblotting indicated a relative reduction in intensity of its correspondent Mr after sAA glycation; and a decrease in its enzymatic activity was observed. Carbonylation levels increased in all glycated samples, except for saliva with fructose. Thiol content decreased only for glycated lysozyme and saliva with MGO. Therefore, glycation of salivary fluid and sAA may have the potential to identify products derived by glycation process. This opens perspectives for further studies on the use of saliva, an easy and non-invasive collection fluid, to monitor glycated proteins in the aging process and evolution of diseases.
Assuntos
Frutose/análise , Produtos Finais de Glicação Avançada/metabolismo , Aldeído Pirúvico/análise , Adulto , Albuminas/análise , Albuminas/química , Feminino , Produtos Finais de Glicação Avançada/análise , Glicosilação , Voluntários Saudáveis , Humanos , Masculino , Muramidase/análise , Muramidase/química , Estresse Oxidativo , Saliva/química , Proteínas e Peptídeos Salivares/metabolismo , Espectrometria de FluorescênciaRESUMO
Several applications arise from the confinement of proteins on surfaces because their stability and biological activity are enhanced. It is also known that the way in which a protein adsorbs on the surface is important for its biological function since its active sites should not be obstructed. In this study, the adsorption properties of hen egg-white lysozyme, HEWL, into a negatively charged silica pore is examined by employing a coarse-grained model and constant-pH Monte Carlo simulations. The role of electrostatic interactions is taken into account via including the Debye-Hückel potentials into the Cα structure-based model. We evaluate the effects of pH, salt concentration, and pore radius on the protein preferential orientation and spatial distribution of its residues regarding the pore surface. By mapping the residues that stay closer to the pore surface, we find that the increase of pH leads to orientational changes of the adsorbed protein when the solution pH gets closer to the HEWL isoelectric point. Under these conditions, the pKa shift of these important residues caused by the adsorption into the charged confining surface results in a HEWL charge distribution that stabilizes the adsorption in the observed protein orientation. We compare our observations to the results of the pKa shift for HEWL available in the literature and to some experimental data.
Assuntos
Muramidase/química , Adsorção , Animais , Galinhas , Concentração de Íons de Hidrogênio , Modelos Moleculares , Método de Monte Carlo , Muramidase/metabolismo , PrótonsRESUMO
Protein immobilization by electrostatic adsorption to a support could represent a good option. On the other hand, lysozyme (EC 3.2.1.17) is a little and basic protein. The objective of this work was to test the functionality of the strategy of Rational Design of Immobilized Derivatives for the immobilization by electrostatic adsorption of egg white lysozyme on SP-Sepharose FastFlow support. The RDID1.0 software was used to predict the superficial lysozyme clusters, the electrostatic configuration probability for each cluster, and the theoretical and estimated maximum quantity of protein to be immobilized. In addition, immobilization was performed and the experimental parameter practical maximum quantity of protein to be immobilized and the enzymatic activity of the immobilized derivative were assessed. The estimated maximum quantity of protein to be immobilized (9.49 protein mg/support g) was close to the experimental practical maximum quantity of protein to be immobilized (14.73 ± 0.09 protein mg/support g). The enzymatic activity assay with the chitosan substrate showed the catalytic functionality of the lysozyme-SP-Sepharose immobilized derivative (35.85 ± 3.07 U/support g), which preserved 78% functional activity. The used algorithm to calculate the estimated maximum quantity of protein to be immobilized works for other proteins, porous solid supports and immobilization methods, and this parameter has a high predictive value, useful for obtaining optimum immobilized derivatives. The applied methodology is valid to predict the most probable protein-support configurations and their catalytic competences, which concur with the experimental results. The produced biocatalyst had a high retention of functional activity. This indicates its functionality in enzymatic bioconversion processes.
Assuntos
Algoritmos , Enzimas Imobilizadas/química , Muramidase/química , Software , Eletricidade EstáticaRESUMO
PURPOSE: In deep burns, wound contraction and hypertrophic scar formation can generate functional derangement and debilitation of the affected part. In order to improve the quality of healing in deep second-degree burns, we developed a new treatment in a preclinical model using nanostructured membranes seeded with mesenchymal stem cells (MSCs). METHODS: Membranes were obtained by reconstitution of bacterial cellulose (reconstituted membrane [RM]) and produced by a dry-cast process, then RM was incorporated with 10% tamarind xyloglucan plus gellan gum 1:1 and 10% lysozyme (RMGT-LZ) and with 10% gellan gum and 10% lysozyme (RMG-LZ). Membrane hydrophobic/hydrophilic characteristics were investigated by static/dynamic contact-angle measurements. They were cultivated with MSCs, and cell adhesion, proliferation, and migration capacity was analyzed with MTT assays. Morphological and topographic characteristics were analyzed by scanning electron microscopy. MSC patterns in flow cytometry and differentiation into adipocytes and osteocytes were checked. In vivo assays used RMG-LZ and RMGT-LZ (with and without MSCs) in Rattus norvegicus rats submitted to burn protocol, and histological sections and collagen deposits were analyzed and immunocytochemistry assay performed. RESULTS: In vitro results demonstrated carboxyl and amine groups made the membranes moderately hydrophobic and xyloglucan inclusion decreased wettability, favoring MSC adhesion, proliferation, and differentiation. In vivo, we obtained 40% and 60% reduction in acute/chronic inflammatory infiltrates, 96% decrease in injury area, increased vascular proliferation and collagen deposition, and complete epithelialization after 30 days. MSCs were detected in burned tissue, confirming they had homed and proliferated in vivo. CONCLUSION: Nanostructured cellulose-gellan-xyloglucan-lysozyme dressings, especially when seeded with MSCs, improved deep second-degree burn regeneration.
Assuntos
Bandagens , Queimaduras/terapia , Celulose/química , Glucanos/química , Células-Tronco Mesenquimais/citologia , Muramidase/química , Nanoestruturas/química , Polissacarídeos Bacterianos/química , Xilanos/química , Animais , Vasos Sanguíneos/patologia , Queimaduras/patologia , Adesão Celular , Diferenciação Celular , Proliferação de Células , Celulose/ultraestrutura , Colágeno/metabolismo , Inflamação/patologia , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/ultraestrutura , Nanoestruturas/ultraestrutura , Ratos Wistar , CicatrizaçãoRESUMO
We describe the synthesis of polymer monoliths inside polypropylene tubes from ink pens. These tubes are cheap, chemically stable, and resistant to pressure. UV-initiated grafting with 5 wt% benzophenone in methanol for 20 min activated the internal surface, thus enabling the covalent binding of ethylene glycol dimethacrylate, also via photografting. The pendant vinyl groups attached a poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith prepared via photopolymerization. These tubes measured 100-110 mm long, with 2 mm of internal diameter. The parent monoliths were functionalized with Na2 SO3 or iminodiacetate to produce strong and weak cation exchangers, respectively. The columns exhibited permeabilities varying from 2.7 to 3.3 × 10-13 m2 , which enabled the separation of proteins at 500 µL/min and back pressures <2.8 MPa. Neither structure collapse nor monolith detachment occurred at flow rates as high as 2.0 mL/min, which produced back pressures between 6.9 and 9.0 MPa. The retention times of ovalbumin, ribonuclease A, cytochrome C, and lysozyme in salt gradient at pH 7.0 followed the order of increasing isoelectric points, confirming the cation exchange mechanism. Separation and determination of lysozyme in egg white proved the applicability of the columns to the analysis of complex samples.