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1.
J Chem Theory Comput ; 15(9): 5144-5153, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31411882

RESUMO

Nontargeted parallel cascade selection molecular dynamics (nt-PaCS-MD) is an enhanced conformational sampling method of proteins, which does not rely on knowledge of the target structure. It makes use of cyclic resampling from some relevant initial structures to expand the searched conformational subspace. The efficiency of nt-PaCS-MD depends on the selections of these initial structures. They are usually stochastically occurring perturbed structures at which larger conformation transitions are about to happen. Reliable identification of these is the key to using nt-PaCS-MD. Two new parameters, the moving root-mean-square deviation (mRMSD) and the inner products of the backbone dihedral angles Φ and Ψ, are introduced as indicators of conformational outliers in MD trajectories. Both are based on the analysis of a time-localized set of coordinates, overcoming the need for a target structure while still capturing the complexity of the conformational transition. The reference to which the mRMSD relates is the close surrounding of the i-th conformation, often the (i-1)st one. Hence the name "time-localized" analysis. In this work, we focus on its interplay with nt-PaCS-MD and show that it increases its effectiveness compared to older versions. The target system is the midsized protein T4 lysozyme (in explicit water) on which we demonstrate the open-closed transition without referring to any target configuration. Additionally, we show that the short MD trajectories can be used for the construction of a free energy landscape of the conformational transition based on the Markov state model.


Assuntos
Simulação de Dinâmica Molecular , Muramidase/química , Bacteriófago T4/enzimologia , Muramidase/metabolismo , Conformação Proteica , Fatores de Tempo
2.
J Photochem Photobiol B ; 197: 111540, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31276926

RESUMO

Protein aggregation can lead to several incurable amyloidosis diseases. The full aggregation pathway is not fully understood, creating the need for new methods of studying this important biological phenomenon. Lysozyme is an amyloidogenic protein which is often used as a model protein for studying amyloidosis. This work explores the potential of employing Lysozyme encapsulated gold nanoclusters (Ly-AuNCs) to study the protein's aggregation. The fluorescence emission properties of Ly-AuNCs were studied in the presence of increasing concentrations of native lysozyme and as a function of pH, of relevance in macromolecular crowding and inflammation-triggered aggregation. AuNC fluorescence was observed to both redshift and increase in intensity as pH is increased or when native lysozyme is added to a solution of Ly-AuNCs at pH 3. The long (µs) fluorescence lifetime component of AuNC emission was observed to decrease under both conditions. Interestingly it was found via Time-Resolved Emission Spectra (TRES) that both AuNC fluorescence components increase in intensity and redshift with increasing pH while only the long lifetime component of AuNC was observed to change when adding native lysozyme to solution; indicating that the underlying mechanisms for the changes observed are fundamentally different for each case. It is possible that the sensitivity of Ly-AuNCs to native lysozyme concentration could be utilized to study early-stage aggregation.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Muramidase/química , Animais , Galinhas , Concentração de Íons de Hidrogênio , Muramidase/metabolismo , Agregados Proteicos/fisiologia , Espectrometria de Fluorescência
3.
Bioresour Technol ; 289: 121703, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31271912

RESUMO

Feasibility of combined lysozyme and rhamolipid (RL) pretreatment on the enhancement of excess sludge (ES) hydrolysis and decomposition was assessed in this study. Results showed lysozyme and RL combined treatment could significantly promote ES hydrolysis and decomposition, an additional 1196.9 mg/L soluble chemical oxygen demand (SCOD), 792.5 mg/L protein and 133.5 mg/L polysaccharide were released compared with the sum of sole RL and sole lysozyme treatment at the optimal RL dosage of 0.3 g/gSS and lysozyme dosage of 0.15 g/gSS after 8 h co-digestion. 45.3% bacteria and 84.5% archaea decomposition degree were gained under the combined treatment at the optimal RL dosage. Class Gammaproteobacteria and genus Methanothrix were the predominant bacteria and archaea with the relative abundance of 72.4% and 60.8%, respectively. After the combined pretreatment, ES was beneficial for volatile fatty acids accumulation and acetic acid dependent methane generating inferred from the results of microbial community composition.


Assuntos
Glicolipídeos/metabolismo , Muramidase/metabolismo , Esgotos/microbiologia , Archaea/metabolismo , Bactérias/metabolismo , Análise da Demanda Biológica de Oxigênio , Ácidos Graxos Voláteis/biossíntese , Hidrólise , Metano/metabolismo , Microbiota
4.
Phys Chem Chem Phys ; 21(23): 12649-12666, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31157335

RESUMO

In the proposed work, the complexation of bioactive flavonoid luteolin with hen egg white lysozyme (HEWL) along with its inhibitory influence on HEWL modification has been explored with the help of multi-spectroscopic and computational methods. The binding affinity has been observed to be moderate in nature (in the order of 104 M-1) and the static quenching mechanism was found to be involved in the fluorescence quenching process. The binding constant (Kb) shows a progressive increase with the increase in temperature from (4.075 ± 0.046 × 104 M-1) at 293 K to (6.962 ± 0.024 × 104 M-1) at 313 K under experimental conditions. Spectroscopic measurements along with molecular docking calculations suggest that Trp62 is involved in the binding site of luteolin within the geometry of HEWL. The positive changes in enthalpy (ΔH = +19.99 ± 0.65 kJ mol-1) as well as entropy (ΔS = +156.28 ± 2.00 J K-1 mol-1) are indicative of the presence of hydrophobic forces that stabilize the HEWL-luteolin complex. The micro-environment around the Trp residues showed an increase in hydrophobicity as indicated by synchronous fluorescence (SFS), three dimensional fluorescence (3D) and red edge excitation (REES) studies. The % α-helix of HEWL showed a marked reduction upon binding with luteolin as indicated by circular dichroism (CD) and Fourier-transform infrared spectroscopy (FTIR) studies. Moreover, luteolin is situated at a distance of 4.275 ± 0.004 nm from the binding site as indicated by FRET theory, and the rate of energy transfer kET (0.063 ± 0.004 ns-1) has been observed to be faster than the donor decay rate (1/τD = 0.606 ns-1), which is indicative of the non-radiative energy transfer during complexation. Leaving aside the binding study, luteolin showed promising inhibitory effects towards the d-ribose mediated glycation of HEWL as well as towards HEWL fibrillation as studied by fluorescence emission and imaging studies. Excellent correlation with the experimental observations as well as precise location and dynamics of luteolin within the binding site has been obtained from molecular docking and molecular dynamics simulation studies.


Assuntos
Luteolina/química , Luteolina/farmacologia , Muramidase/química , Muramidase/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Galinhas , Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Estrutura Molecular , Termodinâmica
5.
Talanta ; 202: 1-10, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171157

RESUMO

Aqueous Biphasic Systems (ABSs) based on deep eutectic solvents (DESs) were determined and applied in the extraction of lysozyme from chicken egg white. Tetrabutylammonium bromide (TBAB) and benzyltributylammonium bromide were utilized as hydrogen-bond acceptors to synthesize six kinds of DESs with different carboxylic acids (such as glycolic acid, Gly). The phase-formation ability of these DESs was evaluated by combining several salts. The results revealed that the content of hydrophilic group and the alkyl side chain length of the carboxylic acids played a crucial role in phase separation process, and the introduce of the benzyl group for quaternary ammonium salt had an aptitude to promote two-phase splitting. Then the system comprising [TBAB][Gly] and Na2SO4 was used to appraise the effect of different experimental parameters on the extraction efficiency, including the amount of DES and salt, the temperature, the values of pH and the ionic strength. More than 98% of lysozyme was transferred into the DES-rich phase at the optimum condition. The activity of lysozyme after the process of extraction still retained 91.73% of initial activity, demonstrating high biocompatibility of the studied system. What's more, the proposed method was successfully utilized for the real sample analysis. Finally, UV-vis, FT-IR, circular dichroism spectra, dynamic light scattering and transmission electron microscopy were employed to investigate the extraction mechanism. All of these results verify the excellent feasibility of the proposed system in the analysis of biological samples.


Assuntos
Muramidase/isolamento & purificação , Animais , Galinhas , Clara de Ovo/química , Muramidase/química , Muramidase/metabolismo , Solventes/química , Água/química
6.
J Chem Phys ; 150(22): 221101, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31202231

RESUMO

Searching for reaction pathways describing rare events in large systems presents a long-standing challenge in chemistry and physics. Incorrectly computed reaction pathways result in the degeneracy of microscopic configurations and inability to sample hidden energy barriers. To this aim, we present a general enhanced sampling method to find multiple diverse reaction pathways of ligand unbinding through nonconvex optimization of a loss function describing ligand-protein interactions. The method successfully overcomes large energy barriers using an adaptive bias potential and constructs possible reaction pathways along transient tunnels without the initial guesses of intermediate or final states, requiring crystallographic information only. We examine the method on the T4 lysozyme L99A mutant which is often used as a model system to study ligand binding to proteins, provide a previously unknown reaction pathway, and show that by using the bias potential and the tunnel widths, it is possible to capture heterogeneity of the unbinding mechanisms between the found transient protein tunnels.


Assuntos
Benzeno/metabolismo , Muramidase/metabolismo , Bacteriófago T4/enzimologia , Ligantes , Modelos Químicos , Simulação de Dinâmica Molecular , Muramidase/genética , Mutação , Ligação Proteica
7.
Anal Chim Acta ; 1070: 112-122, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31103164

RESUMO

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has proven to be an effective technique for aptamers selection. In this study, we present an online reaction based convenient single-step CE-SELEX (ssCE-SELEX) mode with human thrombin (H-Thr) as a model target. The selection progress was monitored through bulk Kd analysis, which showed more than a 1000-fold improvement over the initial library after two rounds of selection. Three selected candidate sequences presented high binding affinities against H-Thr with nanomolar (nM) Kd determined by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM, 56.4-177.1 nM) and CE based non-linear fitting (CE-NLF, 98.2-199.7 nM). They also exhibited high specificities towards H-Thr compared with bovine thrombin, IgG, lysozyme, and lactoferrin. Meanwhile, the Kd results by isothermal titration calorimetry (ITC) confirmed the effective CE in measuring the aptamer affinity. In addition, three candidates were applied as aptasensors in the AuNPs based colorimetric assay, which showed visible color change and good linear relationships (R2 > 0.93) with H-Thr concentration. Furthermore, molecular dynamics (MD) simulation was performed to validate the binding of the three candidates with H-Thr by binding sites and binding free energy. The ssCE-SELEX method avoids off-line incubation, saves time and sample, and may provide a universal and convenient method for aptamers selection.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Sistemas On-Line , Técnica de Seleção de Aptâmeros/métodos , Animais , Calorimetria , Bovinos , Eletroforese Capilar , Humanos , Imunoglobulina G/análise , Lactoferrina/análise , Ligantes , Muramidase/análise , Muramidase/metabolismo , Trombina/análise
8.
Talanta ; 200: 57-66, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036225

RESUMO

In our work, aptamers and hemin/G-quadruplex DNAzyme modified sandwich-rod graphene quantum dots @ graphene oxide @ carbon fiber composite (DNAzyme/L-Apt/GQDs@GO@CF) was successfully prepared for sensitive and selective chemiluminescence (CL) detection of lysozyme (LZM). Initially, GQDs@GO@CF was successfully prepared and characterized. Lysozyme aptamers (L-Apt) as a recognition element and hemin/G-quadruplex DNAzyme (DNAzyme) as a catalyst of luminal - H2O2 were modified on the surface of GQDs@GO@CF, sequentially. The immobilization properties of GQDs@GO@CF to L-Apt and the adsorption properties of L-Apt/GQDs@GO@CF to DNAzyme were also researched, respectively. Then, the modified sandwich-rod carbon fiber composite was applied to the construction of CL biosensor for LZM detection. When LZM existed, DNAzyme would be released from the surface of L-Apt/GQDs@GO@CF and catalyzed the reaction of luminal - H2O2. Under optimized conditions, the CL biosensor for LZM detection showed wide linear range of 2.64 × 10-10 to 6.6 × 10-8 g/L and low detection limit of 1.25 × 10-11 g/L (3δ). Finally, the CL biosensor was successfully used for LZM detection in human urine samples and illustrated the potential application in pratical samples.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Carbono/química , DNA Catalítico/química , Hemina/química , Muramidase/análise , Adsorção , DNA Catalítico/metabolismo , Quadruplex G , Luminescência , Muramidase/metabolismo , Propriedades de Superfície
9.
Talanta ; 199: 472-477, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952286

RESUMO

Columns packed with ultrafine particles (e.g. sub-2 µm porous particles) are suitable for high-resolution, high-speed analytical separation of proteins. However, they require very expensive chromatography systems to provide the ultra-high pressure required for carrying out separations using such columns. Also, frictional heating at high pressure could result in peak broadening and on-column protein degradation. In this paper, we discuss the use of nanoparticles, packed in a box-shaped or cuboid packed-bed device having 50 mm length, 5 mm width, and 3 mm bed-height for fast, high-resolution separation of proteins at low pressure. The low bed height allows the separation to be carried out at low pressure while the cuboid device reduces dispersion effects and thereby keeps the resolution high. Two different types of hydroxyapatite nanoparticles, i.e., needle-shaped (about 20 nm × 150 nm) and spherical (<200 nm) ones, were examined. The experimental results showed that while the needle-shaped nanoparticles were suitable for the separation of small proteins such as lysozyme, the spherical nanoparticles were better suited for separation of larger proteins such as bovine serum albumin and monoclonal antibody. The separation of the two proteins could be carried out in less than 2 min at a pressure lower than 0.8 MPa, using inexpensive chromatography devices a7nd systems, and without high pressure related problems such as frictional heating and on-column protein denaturation.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Durapatita/química , Muramidase/isolamento & purificação , Nanopartículas/química , Soroalbumina Bovina/isolamento & purificação , Animais , Anticorpos Monoclonais/química , Bovinos , Muramidase/química , Muramidase/metabolismo , Tamanho da Partícula , Pressão , Soroalbumina Bovina/química , Propriedades de Superfície , Fatores de Tempo
10.
Talanta ; 199: 507-512, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952291

RESUMO

Black phosphorus quantum dots (BPQDs) can react with Ru(bpy)32+ to generate strong anodic electrogenerated chemiluminescence (ECL). However, the instability and the lack of functional groups on BPQDs limit its further application in the fabrication of ECL biosensor. In the present work, uniform BPQDs-styrene-acrylamide (St-AAm) nanospheres (BSAN) are synthesized by encapsulating BPQDs into St-AAm copolymer nanospheres. Sufficient amount of BPQDs can be embedded into nanospheres, and react with Ru(bpy)32+ to generate strong anodic ECL which is comparable to that of pure BPQDs. Amino group of polymer endows BPQDs the ability to connect with DNA, and can be used to fabricate ECL aptasensor for the sensitive detection of lysozyme. The proposed aptasensor shows high sensitivity, good selectivity and stability for the detection of lysozyme in the range of 0.1-100 pg mL-1 with a detection limit of 0.029 pg mL-1 (3σ). The proposed method reveals the promising ECL sensing application of BP nanomaterials in the detection of various proteins.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Medições Luminescentes , Muramidase/análise , Pontos Quânticos/química , Acrilamida/química , Clara de Ovo/química , Muramidase/metabolismo , Nanoestruturas/química , Fósforo/química , Polímeros/química , Estireno/química
11.
Molecules ; 24(7)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939857

RESUMO

Herein, the degradation of low molecular weight chitosan (CS), with 92% degree of deacetylation (DD), and its nanoparticles (NP) has been investigated in 0.2 mg/mL lysozyme solution at 37 °C. The CS nanoparticles were prepared using glutaraldehyde crosslinking of chitosan in a water-in-oil emulsion system. The morphological characterization of CS particles was carried out using scanning electron microscopy (SEM) and Transmission Electron Microscopy (TEM) techniques. Using attenuated total reflectance Fourier transform infrared (ATR-FTIR) and UV-VIS spectroscopy, the structural integrity of CS and its NPs in lysozyme solution were monitored. The CS powder showed characteristic FTIR bands around 1150 cm-1 associated with the glycosidic bridges (C-O-C bonds) before and after lysozyme treatment for 10 weeks, which indicated no CS degradation. The glutaraldehyde crosslinked CS NPs showed very weak bands associated with the glycosidic bonds in lysozyme solution. Interestingly, the UV-VIS spectroscopic data showed some degradation of CS NPs in lysozyme solution. The results of this study indicate that CS with a high DD and its NPs crosslinked with glutaraldehyde were not degradable in lysozyme solution and thus unsuitable for pulmonary drug delivery. Further studies are warranted to understand the complete degradation of CS and its NPs to ensure their application in pulmonary drug delivery.


Assuntos
Quitosana/química , Reagentes para Ligações Cruzadas/química , Sistemas de Liberação de Medicamentos , Glutaral/química , Pulmão/efeitos dos fármacos , Muramidase/metabolismo , Nanopartículas/química , Quitosana/metabolismo , Reagentes para Ligações Cruzadas/metabolismo , Glutaral/metabolismo , Humanos , Técnicas In Vitro , Nanopartículas/administração & dosagem
12.
Med Sci Monit ; 25: 2658-2671, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30973161

RESUMO

BACKGROUND To fabricate strontium (Sr)-incorporated titanium (Ti) surfaces by a novel 1-step phase-transited lysozyme (PTL) treatment, and investigate the effects of the prepared samples on osteogenesis and osteoimmunoregulation. MATERIAL AND METHODS Five groups of titanium specimens were prepared, including Ti, PTL, PTL@10Sr (PTL coating with 10 mg/mL Sr), PTL@20Sr PTL coating with 20 mg/mL Sr), and PTL@50Sr (PTL coating with 50 mg/mL Sr) groups. Behaviors of bone marrow mesenchymal stem cells (BMSCs) such as initial attachment, spread, proliferation, and migration, on different surfaces were examined by immunofluorescence, MTS assay, and Transwell system. Then the osteogenic differentiation of BMSCs was detected. When an immune response was factored in, the polarization of macrophages induced by the prepared surfaces was detected by real-time PCR, and the response of BMSCs to macrophage-conditioned medium was assessed in terms of cell migration and osteogenic differentiation. Finally, an in vivo study was performed, using the rat femora implant model, to evaluate the potential for osteogenic induction and osteoimmunoregulation of materials. RESULTS Our in vitro experiments indicated that PTL coating could improve cell spread and adhesion, and the stable Sr release of PTL@Sr layers could promote cell migration and osteogenesis. Moreover, PTL@Sr surface could regulate the immune response of macrophages resulting in enhanced BMSCs recruitment and osteogenic differentiation. The in vivo evaluation showed less inflammatory infiltration and improved bone formation in the PTL@20Sr group. CONCLUSIONS The Sr-loaded PTL layers have greater potential for the induction of osteogenic differentiation of BMSCs, meanwhile Sr-loaded PTL layers could adjust the immune response and thus promote osteogenesis both in vitro and in vivo.


Assuntos
Materiais Revestidos Biocompatíveis/farmacologia , Imunomodulação/efeitos dos fármacos , Muramidase/metabolismo , Osteogênese/efeitos dos fármacos , Transição de Fase , Estrôncio/farmacologia , Titânio/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Íons , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Espectroscopia Fotoeletrônica , Células RAW 264.7 , Ratos Sprague-Dawley , Propriedades de Superfície
13.
Chemphyschem ; 20(11): 1456-1466, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30945450

RESUMO

The molecular behaviors of proteins under crowding conditions are crucial for understanding the protein actions in intracellular environments. Under a crowded environment, the distance between protein molecules is almost the same size as the molecular level, thus, both the excluded volume effect and short ranged soft chemical interaction on protein surface could induce the complicated influence on the protein behavior cooperatively. Recently, various kinds of analytical approaches from macroscopic to microscopic aspects have been made to evaluate the crowding effect. The method, however, has not been established to evaluate the surface specific interactions on protein surface. In this study, the analytical method to evaluate the crowding effect has been suggested by using a charge-transfer fluorescence probe, ANS. By employing the unique property of ANS attaching to charged residues on the surface of lysozyme, the crowding effect was focused, while the case was compared as a reference, in which ANS is confined in hydrophobic pockets of BSA. Consequently, the surface specific changes of fluorescence spectra were readily observed under the crowded environment, whereas the fluorescence spectra of ANS in protein inside did not change. This result suggests the fluorescence spectra of ANS binding to protein surface have the capability to estimate the crowding effect of proteins.


Assuntos
Naftalenossulfonato de Anilina/química , Corantes Fluorescentes/química , Muramidase/química , Soroalbumina Bovina/química , Naftalenossulfonato de Anilina/metabolismo , Animais , Bovinos , Galinhas , Fluorescência , Corantes Fluorescentes/metabolismo , Muramidase/metabolismo , Ligação Proteica , Soroalbumina Bovina/metabolismo , Espectrometria de Fluorescência , Eletricidade Estática , Viscosidade
14.
Appl Biochem Biotechnol ; 189(1): 305-317, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30980288

RESUMO

The effect of 16 amino acids (AA) with various physicochemical properties was investigated on human lysozyme (HL) heat-induced amorphous aggregation. UV-Visible spectrophotometry was used to monitor the kinetics of aggregation in the absence and presence of AA, and transmission electron microscopy (TEM) images were taken from the aggregates. To conduct in silico experiments, Autodock vina was used for docking of AA into protein (via YASARA interface), and FTmap information was checked for an insight onto putative binding sites. Prediction of aggregation-prone regions of lysozyme was made by AGGRESCAN and Tango. Among all tested AA, phenylalanine had the best anti-aggregation effect, followed by lysine. In addition, based on in silico tests, Trp 109 and Val 110 of lysozyme are suggested to be of importance in the aggregation process of the enzyme. In conclusion, phenylalanine, arginine, and lysine were found to affect the nucleation phase of lysozyme aggregation and could be considered as suitable stabilizing structures for this enzyme.


Assuntos
Aminoácidos/administração & dosagem , Muramidase/metabolismo , Simulação por Computador , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Transmissão , Espectrofotometria Ultravioleta
15.
J Photochem Photobiol B ; 193: 89-99, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30825814

RESUMO

Binding interactions between the drug Juglone (JUG) and Lysozyme (LYZ) have been explored in details from spectroscopic studies aided by in silico calculations. UV-Vis, steady state and time resolved fluorescence spectroscopic studies indicate the formation of LYZ-JUG complex in the ground state. Quenching of corrected fluorescence spectra of LYZ in presence of JUG at varied concentrations in different temperature range have been estimated from Stern-Volmer (SV) plots. Time resolved fluorescence spectroscopic studies confirm the mechanism of quenching to be of static type. Binding constant associated with the LYZ-JUG complex has been estimated from Scatchard plot. The number of binding sites, thermodynamic parameters and the modes of interaction are also estimated. Synchronous fluorescence spectra monitored at two discrete wavelength windows confirm the prominent role of Tryptophan residues towards quenching of fluorescence in LYZ. The circular dichroism (CD) spectra signify alterations in the population of α-helical content within the secondary structure of LYZ in presence of JUG molecules. REES of LYZ in the presence of JUG further signify definite impact of the drug JUG molecule on the Trp residues of the protein. The experimental observations are supported by in silico molecular docking and molecular dynamics simulations.


Assuntos
Muramidase/metabolismo , Naftoquinonas/metabolismo , Sítios de Ligação , Dicroísmo Circular , Ligações de Hidrogênio , Simulação de Acoplamento Molecular , Muramidase/química , Naftoquinonas/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Espectrometria de Fluorescência , Temperatura Ambiente , Termodinâmica
16.
J Chromatogr A ; 1592: 192-196, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-30871756

RESUMO

Here, a site-specific immobilization strategy for lysozyme was discussed. First, we calculated the surface micro-environment of lysozyme as a model protein and forecasted the nucleophilic attack activity order of six lysine residues within lysozyme, namely, K96> K97 and K33>K1, K13, and K116. Second, lysozyme was immobilized on agarose resin (epoxy group density 22.34 µmol/g) and incubated at pH 9.5 for 12 h. We then compared the peptide maps of free and immobilized lysozyme and established a quantitative detection method for immobilization sites. Third, we studied the effect of immobilization conditions such as epoxy group density and pH upon immobilization sites. When lysozyme was immobilized upon resin with a high epoxy group density (22.34 µmol/g) at pH 9.5 for 12 h, multiple lysozyme immobilization sites were found, including K33, K96 and K97; when lysozyme was immobilized upon resin with a low epoxy group density (11.36 µmol/g) at pH 9.5 for 12 h, only one lysozyme immobilization site was found, K96. The pH also had an effect upon immobilization sites. When immobilization was performed at pH≥10.5 with low epoxy group density resin (11.36 µmol/g) for 12 h, the immobilization sites included at least K33, K96 and K97; when immobilization was performed at pH 9.5 with other conditions remaining unchanged, the only lysozyme immobilization site found was at K96. These experimental results were highly consistent with the forecasted results, revealing some regularities of immobilization site control for lysozyme upon affinity chromatography resin.


Assuntos
Cromatografia de Afinidade , Enzimas Imobilizadas/química , Lisina/metabolismo , Muramidase/metabolismo , Concentração de Íons de Hidrogênio , Sefarose/química
17.
ACS Appl Mater Interfaces ; 11(12): 12133-12141, 2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30839195

RESUMO

Biocatalysis of large-sized substrates finds wide applications. Immobilizing the involved enzymes on solid supports improves biocatalysis yet faces challenges such as enzyme structural perturbation, leaching, and low cost-efficiencies, depending on immobilization strategies/matrices. Carbon nanotubes (CNTs) are attractive matrices but challenged by enzyme leaching (physical adsorption) or perturbation (covalent linking). Zeolitic imidazolate frameworks (ZIFs) overcome these issues. However, our recent study [ J. Am. Chem. Soc., 2018, 140, 16032-16036] showed reduced cost-efficiency as enzymes trapped below the ZIF surfaces cannot participate in biocatalysis; the enzyme-ZIF composites are also unstable under acidic conditions. In this work, we demonstrate the feasibility of using ZIFs to immobilize enzymes on CNT surfaces on two model enzymes, T4 lysozyme and amylase, both of which showed negligible leaching and retained catalytic activity under neutral and acidic conditions. To better understand the behavior of enzymes on CNTs and CNT-ZIF, we characterized enzyme orientation on both matrices using site-directed spin-labeling (SDSL)-electron paramagnetic resonance (EPR), which is immune to the complexities caused by CNT and ZIF background signals and enzyme-matrix interactions. Our structural investigations showed enhanced enzyme exposure to the solvent compared to enzymes in ZIFs alone; orientation of enzymes in matrices itself is directly related to substrate accessibility and, therefore, essential for understanding and improving catalytic efficiency. To the best of our knowledge, this is the first time ZIFs and one-pot synthesis are employed to anchor large-substrate enzymes on CNT surfaces for biocatalysis. This is also the first report of enzyme orientation on the CNT surface and upon trapping in CNT-ZIF composites. Our results are essential for guiding the rational design of CNT-ZIF combinations to improve enzyme stabilization, loading capacity, and catalytic efficiency.


Assuntos
Amilases/metabolismo , Estruturas Metalorgânicas/química , Muramidase/metabolismo , Nanotubos de Carbono/química , Amilases/química , Bacteriófago T4/enzimologia , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Muramidase/química , Marcadores de Spin , Zeolitas/química
18.
J Colloid Interface Sci ; 546: 312-323, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30927595

RESUMO

An electrostatic nanocomplex between naturally occurring ε-poly-l-lysine (εPL) and ß-cyclodextrin sulphate (sCD) was designed, and its capacity to entrap four model proteins with high or low molecular weight and isoelectric point, i.e., lactoferrin, albumin, actinidin, and lysozyme, was investigated. The optimal formulations gave nanocomplexes with an average diameter around 276 ±â€¯16 nm, a ζ-potential of -39 ±â€¯1.5 mV, and a spherical shape with a core-shell structure. Different strategies were pursued to increase the entrapment efficiency for selected proteins, which led to 40-100% entrapment depending on the protein type. Under simulated gastric conditions with pepsin, the complexes protected lactoferrin and albumin against proteolysis, whereas actinidin and lysozyme were intrinsically stable. In Caco-2 cells, these complexes transiently decreased the trans-epithelial electrical resistance, indicating the potential to enhance the paracellular permeability of bioactive macromolecules. Thus, these εPL-sCD complexes would be a promising system for loading diverse proteins for gastric protection and enhancing intestinal absorption.


Assuntos
Albuminas/metabolismo , Cisteína Endopeptidases/metabolismo , Sistemas de Liberação de Medicamentos , Trato Gastrointestinal/efeitos dos fármacos , Lactoferrina/metabolismo , Muramidase/metabolismo , Polilisina/farmacologia , Substâncias Protetoras/farmacologia , beta-Ciclodextrinas/farmacologia , Albuminas/análise , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cisteína Endopeptidases/análise , Relação Dose-Resposta a Droga , Humanos , Lactoferrina/análise , Estrutura Molecular , Muramidase/análise , Tamanho da Partícula , Polilisina/química , Substâncias Protetoras/química , Relação Estrutura-Atividade , Propriedades de Superfície , beta-Ciclodextrinas/química
19.
Proc Natl Acad Sci U S A ; 116(13): 6081-6090, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30846556

RESUMO

The stability of proteins influences their tendency to aggregate, undergo degradation, or become modified in cells. Despite their significance to understanding protein folding and function, quantitative analyses of thermodynamic stabilities have been mostly limited to soluble proteins in purified systems. We have used a highly multiplexed proteomics approach, based on analyses of methionine oxidation rates, to quantify stabilities of ∼10,000 unique regions within ∼3,000 proteins in human cell extracts. The data identify lysosomal and extracellular proteins as the most stable ontological subsets of the proteome. We show that the stability of proteins impacts their tendency to become oxidized and is globally altered by the osmolyte trimethylamine N-oxide (TMAO). We also show that most proteins designated as intrinsically disordered retain their unfolded structure in the complex environment of the cell. Together, the data provide a census of the stability of the human proteome and validate a methodology for global quantitation of folding thermodynamics.


Assuntos
Metionina/metabolismo , Dobramento de Proteína , Estabilidade Proteica , Proteínas/química , Proteoma/metabolismo , Fibroblastos/metabolismo , Humanos , Espectrometria de Massas , Muramidase/metabolismo , Oxirredução , Conformação Proteica , Termodinâmica
20.
Mol Cells ; 42(3): 262-269, 2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30841024

RESUMO

The porcine myeloid antimicrobial peptide (PMAP), one of the cathelicidin family members, contains small cationic peptides with amphipathic properties. We used a putative lysozyme originated from the bacteriophage P22 (P22 lysozyme) as a fusion partner, which was connected to the N-terminus of the PMAP36 peptide, to markedly increase the expression levels of recombinant PMAP36. The PMAP36-P22 lysozyme fusion protein with high solubility was produced in Escherichia coli. The final purified yield was approximately 1.8 mg/L. The purified PMAP36-P22 lysozyme fusion protein exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria (Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Bacillus subtilis). Furthermore, we estimated its hemolytic activity against pig erythrocytes as 6% at the high concentration (128 µM) of the PMAP36-P22 lysozyme fusion protein. Compared with the PMAP36 peptide (12%), our fusion protein exhibited half of the hemolytic activity. Overall, our recombinant PMAP36-P22 lysozyme fusion protein sustained the antimicrobial activity with the lower hemolytic activity associated with the synthetic PMAP36 peptide. This study suggests that the PMAP36-P22 lysozyme fusion system could be a crucial addition to the plethora of novel antimicrobials.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Muramidase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Bactérias/efeitos dos fármacos , Bactérias/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Feminino , Hemólise/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Muramidase/química , Plasmídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Suínos
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