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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.
Phys Chem Chem Phys ; 21(33): 18149-18160, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31389436

RESUMO

Conformational entropies are of great interest when studying the binding of small ligands to proteins or the interaction of proteins. Unfortunately, there are no experimental methods available to measure conformational entropies of all groups in a protein. Instead, they are normally estimated from molecular dynamics (MD) simulations, although such methods show problems with convergence and correlation of motions, and depend on the accuracy of the underlying potential-energy function. Crystallographic atomic displacement parameters (also known as B-factors) are available in all crystal structures and contain information about the atomic fluctuations, which can be converted to entropies. We have studied whether B-factors can be employed to extract conformational entropies for proteins by comparing such entropies to those measured by NMR relaxation experiments or obtained from MD simulations in solution or in the crystal. Unfortunately, our results show that B-factor entropies are unreliable, because they include the movement and rotation of the entire protein, they exclude correlation of the movements and they include contributions other than the fluctuations, e.g. static disorder, as well as errors in the model and the scattering factors. We have tried to reduce the first problem by employing translation-libration-screw refinement, the second by employing a description of the correlated movement from MD simulations, and the third by studying only the change in entropy when a pair of ligands binds to the same protein, thoroughly re-refining the structures in exactly the same way and using the same set of alternative conformations. However, the experimental B-factors seem to be incompatible with fluctuations from MD simulations and the precision is too poor to give any reliable entropies.


Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Temperatura Ambiente , Cristalografia por Raios X , Entropia , Galectina 3/química , Ligantes , Muramidase/química , Conformação Proteica , Tripsina/química
3.
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
4.
J Chem Phys ; 151(1): 015101, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31272172

RESUMO

We use extended depolarized light scattering spectroscopy to study the dynamics of water in a lysozyme-trehalose aqueous solution over a broad time scale, from hundreds to fractions of picoseconds. We provide experimental evidence that the sugar, present in the ternary solution in quantity relevant for biopreservation, strongly modifies the solvation properties of the protein. By comparing aqueous solutions of lysozyme with and without trehalose, we show that the combined action of sugar and protein produces an exceptional dynamic slowdown of a fraction of water molecules around the protein, which become more than twice slower than in the absence of trehalose. We speculate that this ultraslow water may be caged between the sugar and protein surface, consistently with a water entrapment scenario. We also demonstrate that the dynamics of these water molecules gets slower and slower upon cooling. On the basis of these findings, we believe such ultraslow water close to the lysozyme is likely to be involved in the mechanism of bioprotection.


Assuntos
Luz , Muramidase/química , Espalhamento de Radiação , Análise Espectral/métodos , Trealose/química , Água/química
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.
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
8.
Carbohydr Polym ; 216: 107-112, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31047046

RESUMO

A simple, convenient and inexpensive method for the preparation of magnetic chitin composite, in which magnetite particles are densely covered with the polysaccharide shell has been developed. Two-step procedure for magnetic chitin preparation includes: (i) adsorption of chitosan onto magnetite particles and (ii) N-selective acetylation of chitosan to produce magnetic chitin. The composite combines the magnetic properties of magnetite and the adsorption properties of chitin. The synthesized magnetic chitin is an efficient adsorbent of ß-d-GlcNAc-specific lectins and lysozyme. The adsorption capacity of magnetic chitin for wheat germ (Triticum vulgaris) and potato (Solanum tuberosum) lectins was in the range of 67-86 mg g-1 of the adsorbent. Magnetic chitin showed the high capacity for enzyme-lysozyme. Synthesized adsorbent is environmentally friendly and recyclable. Magnetic chitin may be used as a promising multi-purpose adsorbent of pollutants of organic or inorganic nature.


Assuntos
Quitina/química , Nanopartículas de Magnetita/química , Muramidase/química , Soroalbumina Bovina/química , Aglutininas do Germe de Trigo/química , Adsorção , Animais , Bovinos , Galinhas , Quitina/síntese química , Química Verde/métodos , Fenômenos Magnéticos , Solanum tuberosum/química , Triticum/química
9.
Carbohydr Polym ; 216: 25-35, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31047065

RESUMO

The unique physicochemical and functional characteristics of starch-based biomaterials and wound dressings have been proposed for several biomedical applications. Film dressings of cornstarch/hyaluronic acid/ ethanolic extract of propolis (CS/HA/EEP) were prepared by solvent-casting and characterized by attenuated total reflectance/Fourier transform infrared spectroscopy, scanning electron microscopy, gas chromatography/mass spectrometry, light transmission, opacity measurements, EEP release, equilibrium swelling, and in vitro and in vivo evaluations. The CS/HA/0.5%EEP film dressing exhibited higher antibacterial activity against Staphylococcus aureus (2.08 ± 0.14 mm), Escherichia coli (2.64 ± 0.18 mm), and Staphylococcus epidermidis (1.02 ± 0.15 mm) in comparison with the CS, CS/HA, and CS/HA/0.25%EEP films. Also, it showed no cytotoxicity for the L929 fibroblast cells. This wound dressing could effectively accelerate the wound healing process at Wistar rats' skin excisions. These results indicate that enrichment of cornstarch wound dressings with HA and EEP can significantly enhance their potential efficacy as wound dressing material.


Assuntos
Antibacterianos/farmacologia , Ácido Hialurônico/química , Curativos Oclusivos , Própole/farmacologia , Amido/química , Animais , Antibacterianos/química , Antibacterianos/toxicidade , Galinhas , Escherichia coli/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Ácido Hialurônico/toxicidade , Hidrólise , Muramidase/química , Própole/química , Própole/toxicidade , Ratos Wistar , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Amido/toxicidade , Cicatrização/efeitos dos fármacos
10.
Carbohydr Polym ; 217: 160-167, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31079673

RESUMO

Polysaccharides can be modified by reactive functional groups to enable chemical crosslinking. We studied how different methods of crosslinking methacrylate-functionalized chitosan affected the network structures and various properties relevant for utilization of the chemically crosslinked hydrogels in biomedical applications, including tissue engineering and delivery of therapeutic agents. Four chitosan hydrogels were made by either the free radical polymerization with varying initiation kinetics and an addition of chain transfer agents or the based-catalyzed Michael-type addition reaction. Four chitosan hydrogels having identical polymer fractions at equilibrium swelling exhibited marked differences in shear moduli, dextran diffusion rate, and especially enzymatic degradation behaviors. Hydrogels made by the free radical polymerization with no chain transfer agent were highly resistant to complete degradation by enzyme for an extended period. We inferred that such resistance originated from chain bundles characterized by densely branched networks of chitosan chains, which was determined by small-angle X-ray scattering analysis.


Assuntos
Quitosana/química , Hidrogéis/química , Quitosana/síntese química , Reagentes para Ligações Cruzadas/síntese química , Reagentes para Ligações Cruzadas/química , Dextranos/química , Difusão , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Humanos , Hidrogéis/síntese química , Hidrólise , Metacrilatos/síntese química , Metacrilatos/química , Muramidase/química , Polimerização
11.
Pharm Res ; 36(7): 107, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31111248

RESUMO

PURPOSE: To provide new insights into how protein-surfactant competitive adsorbtion and corresponding surface tension reduction properties at the air-water and oil-water interface are impacted by the type of protein and the associated protein surface rheology. METHOD: Interfacial Rheology was utilized to obtain surface G' and G" as a function of frequency. Force tensiometry was utilized to obtain changes in surface tension as a function of surfactant concentration. The impact on surface properties of two different proteins i.e. BSA and Lysozyme was investigated as a function of surfactant concentration i.e. polysorbates PS 20, PS 80 and Poloxomer (Kolliphor P188). RESULTS: Surface tension and interfacial tension measurements for BSA showed that in mixed BSA/polysorbate surfactant systems, BSA dominates the interfacial behavior at both the air-water and oil-water interfaces, until a high polysorbate concentration of 0.1 mg/ml. At these high polysorbate concentrations a mixed BSA-Polysorbate interfacial layer is formed as corroborated by the surface elasticity values being lower than that of pure BSA but higher than that of pure Polysorbate. For Kolliphor, it was observed that Kolliphor was unable to displace BSA at any concentration. This is corroborated by the high surface elasticity of the BSA which is maintained in the presence of Kolliphor. Surface and interfacial tension measurements for lysozyme show that for mixed lysozyme/polysorbate surfactant systems, the surface tension values are lower than that exhibited by either the lysozyme or the polysorbate surfactants. This potentially indicates the formation of a mixed layer of lysozyme and polysorbate. At the high polysorbate concentrations probed, the surface elasticity values are however closer to that of pure polysorbates, indicating that the mixed layer may be more heavily polysorbate dominated, especially at high polysorbate concentrations. For Kolliphor, the response was similar to that seen in the Kolliphor-BSA system in which the Kolliphor was not able to displace the protein i.e. Lysozyme. CONCLUSIONS: In conclusion, it was seen that competitive adsorption between proteins and common excipient surfactants is dictated by the type of protein and its effective structuring/rigidity at the surface as reflected through surface elasticity and surface tan delta values. BSA was seen to exhibit a higher surface elasticity than lysozyme, and therefore has a more rigid structure and is more competitive at the interface.


Assuntos
Muramidase/química , Poloxâmero/química , Polissorbatos/química , Soroalbumina Bovina/química , Tensoativos/química , Adsorção , Elasticidade , Excipientes , Polietilenoglicóis , Reologia/métodos , Estearatos , Propriedades de Superfície , Tensão Superficial
12.
J Chromatogr A ; 1600: 80-86, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31047663

RESUMO

Lysozyme is widely used in medical, food and industrial fields due to its bacteriolytic effect and thus it is significant to design and develop a specific adsorbent with high adsorption capacity and selectivity for lysozyme. Inspired by the high uptake capacity of tetrafluoroterephthalonitrile-crosslinked porous ß-cyclodextrin polymers (P-CDPs) and the noncovalent interaction between lysozyme and carboxyl groups, the carboxyl-functionalized P-CDPs (P-CDP-COO-) were synthesized by base-catalysed hydrolysis of nitrile group in P-CDPs to carboxyl. Porous structure, large extent of carboxylic functional groups, cyclodextrin's preventing aggregation, and negatively charged make P-CDP-COO- possess an outstanding adsorption capability for lysozyme. The maximum saturated adsorption capacity reaches 1520 mg g-1, which is much better than the parent polymer and other reported materials. The as-prepared material was successfully utilized for the selective extraction of lysozyme from egg white.


Assuntos
Técnicas de Química Analítica/métodos , Clara de Ovo/química , Muramidase/isolamento & purificação , beta-Ciclodextrinas/química , Adsorção , Ácidos Carboxílicos/química , Ciclodextrinas/química , Muramidase/química , Porosidade
13.
J Chromatogr A ; 1599: 55-65, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31036361

RESUMO

Peak broadening in small columns is dominated by spreading in the extra column volume and not by hydrodynamic dispersion or mass transfer resistances. Computational fluid dynamics (CFD) permits to study the influence of these effects separately. Here, peak broadening of three single component solutes - silica nanoparticles, acetone, and lysozyme - was experimentally determined for two different columns (100 mm × 8 mm inner diameter and 10 mm × 5 mm inner diameter) under non-binding conditions. A mass transfer model between mobile and stationary phases as well as a hydrodynamic dispersion model were implemented in the CFD environment STAR-CCM+®. The mass transfer model combines a model of external mass transfer with a model of pore diffusion. The model was validated with experiments performed on the larger column. We find that extra column volume plays an important role in peak broadening of the silica nanoparticles pulse in that column; it is less important for acetone and is weakly pronounced for lysozyme. Hydrodynamic dispersion plays the dominant role at low and medium flow rates for acetone because we are in a regime of 1-10 ReSc. Mass transfer is important for high flow rates of acetone and for all flow rates of lysozyme. Then, peak broadening was predicted in the smaller column with the packed bed parameters taken from larger column. The scalability of the prepacked columns is demonstrated for acetone and silica nanoparticles by excellent agreement with the experimental data. In contrast to the larger column, peak broadening in the smaller column is dominated by extra column volume for all solutes. Peak broadening of lysozyme is controlled only at high flow rates by mass transfer and overrides extra column volume and hydrodynamic dispersion. CFD simulations with implemented mass transfer models successfully model peak broadening in chromatography columns taking all broadening effects into consideration and therefore are a valuable tool for scale up and scale down. Our simulations underscore the importance of extra column volume.


Assuntos
Cromatografia , Modelos Químicos , Acetona/química , Simulação por Computador , Difusão , Muramidase/química , Dióxido de Silício/química
14.
J Phys Chem Lett ; 10(11): 3051-3056, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31081645

RESUMO

Despite the importance of protein dynamics to function, studying exchange between multiple conformational states remains a challenge because sparsely populated states are invisible to conventional techniques. CEST NMR experiments can detect minor states with lifetimes between 5 and 200 ms populated to a level of just ∼1%. However, CEST often cannot provide the exchange mechanism for processes involving three or more states, leaving the role of the detected minor states unknown. Here a double-resonance CEST experiment to determine the kinetics of multistate exchange is presented. The approach that involves irradiating resonances from two minor states simultaneously is used to study the exchange of T4 lysozyme (T4L) between the dominant native state and two minor states, the unfolded state and a second minor state (B), each populated to only ∼4%. Regular CEST does not provide the folding mechanism, but double-resonance CEST clearly shows that T4L can fold directly without going through B.


Assuntos
Muramidase/química , Simulação por Computador , Cinética , Espectroscopia de Ressonância Magnética , Modelos Teóricos , Conformação Proteica , Dobramento de Proteína
15.
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
16.
Chemistry ; 25(31): 7501-7514, 2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-30958585

RESUMO

Nanoparticles have great potential to be used in various biomedical applications, including therapy or diagnosis of amyloid-related diseases. The physical and chemical properties of iron oxide superparamagnetic nanoparticles (MNPs) functionalized with different amino acids (AAs), namely, with lysine (Lys), glycine (Gly), or tryptophan (Trp), have been characterized. The cytotoxicity of nanoparticles and their effect on amyloid fibrillization of lysozymes in vitro was also verified. The AA-MNPs under study are nontoxic to human SHSY5Y neuroblastoma cells. Moreover, the AA-MNPs were able to significantly inhibit lysozyme amyloid fibrillization and destroy amyloid fibrils. Kinetic studies revealed that the presence of AA-MNPs affected lysozyme fibrillization, namely, the lag phase and steady-state phase of the growth curves. The most effective activities were observed for Trp-MNPs, which revealed the importance of aromatic rings in the structure of AAs used as coating agents. The obtained results indicate the possible application of these AA-MNPs in the treatment of amyloid diseases associated with lysozyme or other amyloidogenic proteins.


Assuntos
Aminoácidos/química , Aminoácidos/farmacologia , Amiloide/antagonistas & inibidores , Amiloide/química , Nanopartículas de Magnetita/química , Muramidase/química , Agregados Proteicos/efeitos dos fármacos , Amiloide/ultraestrutura , Animais , Linhagem Celular Tumoral , Galinhas , Humanos , Cinética , Nanopartículas de Magnetita/ultraestrutura , Modelos Moleculares , Muramidase/ultraestrutura , Conformação Proteica/efeitos dos fármacos
17.
Fish Shellfish Immunol ; 89: 198-206, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30946959

RESUMO

Lysozymes are important immune effectors present in phylogenetically diverse organisms. They play vital roles in bacterial elimination during early immune responses. In the present study, a second invertebrate-type (i-type) lysozyme gene from razor clam Sinonovacula constricta (denoted as ScLYZ-2) was cloned by RACE and nested PCR methods. The full-length cDNA sequences of ScLYZ-2 were 1558 bp, including a 5' untranslated region (UTR) of 375 bp, an open reading frame of 426 bp, and a 3'-UTR of 757 bp with polyadenylation signal sequence (AATAAA) located upstream of the poly(A) tail. SMART analysis showed that ScLYZ-2 contains a signal peptide in the first 16 amino acid (AA) sequences and a destabilase domain located from 24 to 134 AA sequences. The deduced AA sequences of ScLYZ-2 were highly similar (42%-58%) to other known lysozyme genes of bivalve species. Multiple alignments of AA sequences showed that ScLYZ-2 possesses the classical i-type lysozyme family signature of two motifs ["MDVGSLSCGP(Y/F)QIK" and "CL(E/L/R/H)C(I/M)C"] and two catalytic residues (Glu35 and Asp46). Moreover, phylogenetic analysis showed that ScLYZ-2 is a new member of the i-type lysozyme family. In healthy razor clams, ScLYZ-2 was highly expressed in the hepatopancreas, followed by the gills, water pipes, and abdominal foot. Lysozyme activity and ScLYZ-2 expression levels were significantly upregulated in the hepatopancreas and gills after being infected with V. splendidus, V. harveyi, V. parahaemolyticus and S. aureus and M. luteus. Moreover, the recombinant ScLYZ-2 had strong antimicrobial activities against V. splendidus, V. harveyi, and V. parahaemolyticus. Furthermore, the minimal inhibitory concentration of the recombinant ScLYZ-2 against V. parahaemolyticus was 7.2 µmol/mL. Taken together, our results show that ScLYZ-2 plays an important role in the immune defense of razor clam by eliminating pathogenic microorganisms.


Assuntos
Bivalves/genética , Bivalves/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Muramidase/genética , Muramidase/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bivalves/enzimologia , Perfilação da Expressão Gênica , Micrococcus luteus/fisiologia , Muramidase/química , Filogenia , Alinhamento de Sequência , Staphylococcus aureus/fisiologia , Vibrio/fisiologia
18.
Se Pu ; 37(4): 392-397, 2019 Apr 08.
Artigo em Chinês | MEDLINE | ID: mdl-30977341

RESUMO

In this work, a novel polymer membrane molecularly imprinted on a silanized glass substrate was prepared. Lysozyme was chosen as the template protein, and acrylamide and N,N'-methylenebisacrylamide were used as the functional monomer and crosslinking agent, respectively. The adsorption equilibrium time, maximum adsorption capacity, specific recognition ability, reusability, and application of the prepared lysozyme-imprinted polymer membrane were investigated experimentally. The prepared polymer membrane exhibited special recognition ability and could quickly adsorb lysozyme. Under optimal conditions, the adsorption equilibrium time of the prepared polymer membrane for lysozyme was 5 min, and the adsorption behavior conformed to the Langmuir adsorption model; the theoretical maximum adsorption capacity was 42.5 mg/g, and the adsorption capacity for an egg sample was 30 mg/g. Additionally, the polymer membrane showed a high selectivity toward the template protein, lysozyme, and exhibited no obvious adsorption of several interfering proteins; the imprinting factor was 3.0. The maximum adsorption capacity of the polymer membrane decreased by 5% after being reused five times. The polymer membrane was used for the adsorption of lysozyme in samples, and good results were obtained. This strategy has potential for the separation and enrichment of target proteins in complex biological samples.


Assuntos
Membranas Artificiais , Impressão Molecular , Muramidase/química , Polímeros/química , Acrilamida , Adsorção
19.
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
20.
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
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