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1.
Nat Commun ; 11(1): 4817, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968056

RESUMO

Lysozymes are among the best-characterized enzymes, acting upon the cell wall substrate peptidoglycan. Here, examining the invasive bacterial periplasmic predator Bdellovibrio bacteriovorus, we report a diversified lysozyme, DslA, which acts, unusually, upon (GlcNAc-) deacetylated peptidoglycan. B. bacteriovorus are known to deacetylate the peptidoglycan of the prey bacterium, generating an important chemical difference between prey and self walls and implying usage of a putative deacetyl-specific "exit enzyme". DslA performs this role, and ΔDslA strains exhibit a delay in leaving from prey. The structure of DslA reveals a modified lysozyme superfamily fold, with several adaptations. Biochemical assays confirm DslA specificity for deacetylated cell wall, and usage of two glutamate residues for catalysis. Exogenous DslA, added ex vivo, is able to prematurely liberate B. bacteriovorus from prey, part-way through the predatory lifecycle. We define a mechanism for specificity that invokes steric selection, and use the resultant motif to identify wider DslA homologues.


Assuntos
Bdellovibrio bacteriovorus/enzimologia , Bdellovibrio bacteriovorus/metabolismo , Muramidase/química , Muramidase/metabolismo , Periplasma/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bdellovibrio bacteriovorus/genética , Parede Celular , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Muramidase/genética , Mutação , Peptidoglicano/metabolismo , Fenótipo , Conformação Proteica , Especificidade por Substrato
2.
Nat Commun ; 11(1): 3714, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709852

RESUMO

The detailed understanding of the binding of small molecules to proteins is the key for the development of novel drugs or to increase the acceptance of substrates by enzymes. Nowadays, computer-aided design of protein-ligand binding is an important tool to accomplish this task. Current approaches typically rely on high-throughput docking essays or computationally expensive atomistic molecular dynamics simulations. Here, we present an approach to use the recently re-parametrized coarse-grained Martini model to perform unbiased millisecond sampling of protein-ligand interactions of small drug-like molecules. Remarkably, we achieve high accuracy without the need of any a priori knowledge of binding pockets or pathways. Our approach is applied to a range of systems from the well-characterized T4 lysozyme over members of the GPCR family and nuclear receptors to a variety of enzymes. The presented results open the way to high-throughput screening of ligand libraries or protein mutations using the coarse-grained Martini model.


Assuntos
Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas/química , Bacteriófago T4/enzimologia , Biofísica , Biologia Computacional , Ensaios de Triagem em Larga Escala , Ligantes , Simulação de Acoplamento Molecular , Muramidase/química , Conformação Proteica , Termodinâmica
3.
BMC Bioinformatics ; 21(1): 275, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611389

RESUMO

BACKGROUND: Protein engineering has many applications for industry, such as the development of new drugs, vaccines, treatment therapies, food, and biofuel production. A common way to engineer a protein is to perform mutations in functionally essential residues to optimize their function. However, the discovery of beneficial mutations for proteins is a complex task, with a time-consuming and high cost for experimental validation. Hence, computational approaches have been used to propose new insights for experiments narrowing the search space and reducing the costs. RESULTS: In this study, we developed Proteus (an acronym for Protein Engineering Supporter), a new algorithm for proposing mutation pairs in a target 3D structure. These suggestions are based on contacts observed in other known structures from Protein Data Bank (PDB). Proteus' basic assumption is that if a non-interacting pair of amino acid residues in the target structure is exchanged to an interacting pair, this could enhance protein stability. This trade is only allowed if the main-chain conformation of the residues involved in the contact is conserved. Furthermore, no steric impediment is expected between the proposed mutations and the surrounding protein atoms. To evaluate Proteus, we performed two case studies with proteins of industrial interests. In the first case study, we evaluated if the mutations suggested by Proteus for four protein structures enhance the number of inter-residue contacts. Our results suggest that most mutations proposed by Proteus increase the number of interactions into the protein. In the second case study, we used Proteus to suggest mutations for a lysozyme protein. Then, we compared Proteus' outcomes to mutations with available experimental evidence reported in the ProTherm database. Four mutations, in which our results agree with the experimental data, were found. This could be initial evidence that changes in the side-chain of some residues do not cause disturbances that harm protein structure stability. CONCLUSION: We believe that Proteus could be used combined with other methods to give new insights into the rational development of engineered proteins. Proteus user-friendly web-based tool is available at < http://proteus.dcc.ufmg.br >.


Assuntos
Proteínas/química , Interface Usuário-Computador , Algoritmos , Bases de Dados de Proteínas , Muramidase/química , Muramidase/genética , Muramidase/metabolismo , Mutagênese , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo
4.
Int J Nanomedicine ; 15: 4705-4716, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636626

RESUMO

Purpose: Ultra-small gold nanoclusters (AuNCs), as emerging fluorescent nanomaterials with excellent biocompatibility, have been widely investigated for in vivo biomedical applications. However, their effects in guiding osteogenic differentiation have not been investigated, which are important for osteoporosis therapy and bone regeneration. Herein, for the first time, lysozyme-protected AuNCs (Lys-AuNCs) are used to stimulate osteogenic differentiation, which have the potential for the treatment of bone disease. Methods: Proliferation of MC3T3E-1 is important for osteogenic differentiation. First, the proliferation rate of MC3T3E-1 was studied by Cell Counting Kit-8 (CCK8) assays. Signaling pathways of PI3K/Akt play central roles in controlling proliferation throughout the body. The expression of PI3K/Akt was investigated in the presence of lysozyme, and lysozyme-protected AuNCs (Lys-AuNCs) by Western blot (WB) and intracellular cell imaging to evacuate the osteogenic differentiation mechanisms. Moreover, the formation of osteoclasts (OC) plays a negative role in the differentiation of osteoblasts. Nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) signaling pathways are used to understand the negative influence of the osteogenic differentiation by the investigation of Raw 264.7 cell line. Raw 264.7 (murine macrophage-like) cells and NIH/3T3 (mouse fibroblast) cells were treated with tyloxapol, and the cell viability was assessed. Raw 264.7 cells have also been used for in vitro studies, on understanding the osteoclast formation and function. The induced osteoclasts were identified by TRAP confocal fluorescence imaging. These key factors in osteoclast formation, such as (NFATc-1, c-Fos, V-ATPase-2 and CTSK), were investigated by Western blot. Results: Based on the above investigation, Lys-AuNCs were found to promote osteogenic differentiation and decrease osteoclast activity. It is noteworthy that the lysozyme (protected template), AuNPs, or the mixture of Lysozyme and AuNPs have negligible effects on osteoblastic differentiation compared to Lys-AuNCs. Conclusion: This study opens up a novel avenue to develop a new gold nanomaterial for promoting osteogenic differentiation. The possibility of using AuNCs as nanomedicines for the treatment of osteoporosis can be expected.


Assuntos
Nanopartículas Metálicas/química , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Ouro/farmacologia , Nanopartículas Metálicas/administração & dosagem , Camundongos , Muramidase/química , Muramidase/metabolismo , Fatores de Transcrição NFATC/metabolismo , Nanomedicina/métodos , Osteoblastos/citologia , Osteoclastos/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7
5.
Food Chem ; 327: 127038, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32447136

RESUMO

Polyacrylonitrile nanofiber membrane functionalized with tris(hydroxymethyl)aminomethane (P-Tris) was used in affinity membrane chromatography for lysozyme adsorption. The effects of pH and protein concentration on lysozyme adsorption were investigated. Based on Langmuir model, the adsorption capacity of P-Tris nanofiber membrane was estimated to be 345.83 mg/g. For the operation of dynamic membrane chromatography with three-layer P-Tris nanofiber membranes, the optimal operating conditions were at pH 9, 1.0 mL/min of feed flow rate, and 2 mg/mL of feed concentration. Chicken egg white (CEW) was applied as the crude feedstock of lysozyme in the optimized dynamic membrane chromatography. The percent recovery and purification factor of lysozyme obtained from the chromatography were 93.28% and 103.98 folds, respectively. Our findings demonstrated the effectiveness of P-Tris affinity nanofiber membrane for the recovery of lysozyme from complex CEW solution.


Assuntos
Galinhas , Cromatografia de Afinidade/métodos , Clara de Ovo/química , Membranas Artificiais , Muramidase/isolamento & purificação , Nanofibras/química , Trometamina/química , Adsorção , Animais , Muramidase/química
6.
Nucleic Acids Res ; 48(W1): W17-W24, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32343309

RESUMO

PDBMD2CD is a new web server capable of predicting circular dichroism (CD) spectra for multiple protein structures derived from molecular dynamics (MD) simulations, enabling predictions from thousands of protein atomic coordinate files (e.g. MD trajectories) and generating spectra for each of these structures provided by the user. Using MD enables exploration of systems that cannot be monitored by direct experimentation. Validation of MD-derived data from these types of trajectories can be difficult via conventional structure-determining techniques such as crystallography or nuclear magnetic resonance spectroscopy. CD is an experimental technique that can provide protein structure information from such conditions. The website utilizes a much faster (minimum ∼1000×) and more accurate approach for calculating CD spectra than its predecessor, PDB2CD (1). As well as improving on the speed and accuracy of current methods, new analysis tools are provided to cluster predictions or compare them against experimental CD spectra. By identifying a subset of the closest predicted CD spectra derived from PDBMD2CD to an experimental spectrum, the associated cluster of structures could be representative of those found under the conditions in which the MD studies were undertaken, thereby offering an analytical insight into the results. PDBMD2CD is freely available at: https://pdbmd2cd.cryst.bbk.ac.uk.


Assuntos
Dicroísmo Circular/métodos , Simulação de Dinâmica Molecular , Proteínas/química , Software , Muramidase/química , Conformação Proteica , Desdobramento de Proteína
7.
Nat Commun ; 11(1): 1271, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152274

RESUMO

Protein dynamics are integral to biological function, yet few techniques are sensitive to collective atomic motions. A long-standing goal of X-ray crystallography has been to combine structural information from Bragg diffraction with dynamic information contained in the diffuse scattering background. However, the origin of macromolecular diffuse scattering has been poorly understood, limiting its applicability. We present a finely sampled diffuse scattering map from triclinic lysozyme with unprecedented accuracy and detail, clearly resolving both the inter- and intramolecular correlations. These correlations are studied theoretically using both all-atom molecular dynamics and simple vibrational models. Although lattice dynamics reproduce most of the diffuse pattern, protein internal dynamics, which include hinge-bending motions, are needed to explain the short-ranged correlations revealed by Patterson analysis. These insights lay the groundwork for animating crystal structures with biochemically relevant motions.


Assuntos
Movimento (Física) , Muramidase/química , Difração de Raios X , Cristalização , Simulação de Dinâmica Molecular , Fônons
8.
Nat Commun ; 11(1): 1231, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144241

RESUMO

We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.


Assuntos
Muramidase/metabolismo , Proteínas Virais/metabolismo , Bacteriófago T4/enzimologia , Bacteriófago T4/genética , Biocatálise , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Simulação de Dinâmica Molecular , Método de Monte Carlo , Muramidase/química , Muramidase/genética , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas Virais/química , Proteínas Virais/genética
9.
J Food Sci ; 85(4): 1037-1044, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32175601

RESUMO

Effects of lysozyme (LYS) combined with cinnamaldehyde (CA) on quality enhancement of olive flounder (Paralichthys olivaceus) fillets during refrigerated storage at 4 °C for 20 days were assessed. Changes of total viable count (TVC), K-value, total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA), texture profile analysis (TPA), and trichloroacetic acid-soluble peptide (TCA-soluble peptide) in samples were determined periodically. Results demonstrated that the combination of LYS and CA treatment enhanced the antibacterial activity against S. putrefaciens and P. fluorescens, and lowered TVC values. Meanwhile, LYS combined with CA significantly retarded the increases of TBA value, TVB-N, K-value, and TCA-soluble peptide content compared to the control. Furthermore, the combined treatment also effectively maintained the texture properties of flounder fillets during the storage period. The efficiency was better than that of LYS or CA treatment alone. Thus, LYS combined with CA is promising in olive flounder shelf life extension.


Assuntos
Acroleína/análogos & derivados , Produtos Pesqueiros/análise , Conservação de Alimentos/métodos , Muramidase/química , Acroleína/análise , Animais , Linguados , Conservação de Alimentos/instrumentação , Conservantes de Alimentos/análise , Armazenamento de Alimentos , Nitrogênio/análise
10.
Nat Commun ; 11(1): 996, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081905

RESUMO

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.


Assuntos
Cristalografia/métodos , Proteínas/química , Microscopia Eletrônica de Transmissão e Varredura , Modelos Moleculares , Muramidase/química , Muramidase/ultraestrutura , Nanopartículas/química , Nanopartículas/ultraestrutura , Proteínas de Matriz de Corpos de Inclusão/química , Proteínas de Matriz de Corpos de Inclusão/ultraestrutura , Tamanho da Partícula , Conformação Proteica , Proteínas/ultraestrutura
11.
Chemistry ; 26(24): 5500-5507, 2020 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-32092201

RESUMO

Polydopamine (PD) and melanin species are chemically complex systems, the formation and properties of which are incompletely understood. Inspired by the role of functional amyloids in melanin biosynthesis, this paper examines the influences of the supramolecular structure of amyloids on oxidative polymerization of dopamine. Kinetic analyses on the formation of PD species in the presence of hen egg white lysozyme (HEWL) fibers or soluble HEWL revealed that both forms gave rise to the total quantity of PD species, but the rate of their formation could be accelerated only by the amyloid form. PD species formed with HEWL fibers showed a morphology of bundled fibers, whereas those with soluble HEWL had a mesh-like structure. Amyloid fibers of recombinant Pmel17 had properties similar to those of HEWL fibers in modulating PD formation. The results presented here suggest how nature designs functionality with an amyloid structure and can help understand and engineer chemistries of other functional amyloids.


Assuntos
Amiloide/química , Indóis/química , Melaninas/química , Muramidase/química , Polímeros/química , Amiloide/metabolismo , Animais , Cinética , Muramidase/metabolismo
12.
Chemistry ; 26(26): 5799-5809, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32104951

RESUMO

The influence of the composition of chaotropic polyoxometalate (POM) anions on their affinity to biological systems was studied by means of atomistic molecular dynamics (MD) simulations. The variations in the affinity to hen egg-white lysozyme (HEWL) were analyzed along two series of POMs whereby the charge or the size and shape of the metal cluster are modified systematically. Our simulations revealed a quadratic relationship between the charge of the POM and its affinity to HEWL as a consequence of the parabolic growth of POM⋅⋅⋅water interaction with the charge. As the charge increases, POMs become less chaotropic (more kosmotropic) increasing the number and the strength of POM-water hydrogen bonds and structuring the solvation shell around the POM. This atomistic description explains the proportionally larger desolvation energies and less protein affinity for highly charged POMs, and consequently, the preference for moderate charge densities (q/M=0.33). Also, our simulations suggest that POM⋅⋅⋅protein interactions are size-specific. The cationic pockets of HEWL protein show a preference for Keggin-like structures, which display the optimal dimensions (≈1 nm). Finally, we developed a quantitative multidimensional model for protein affinity with predictive ability (r2 =0.97; q2 =0.88) using two molecular descriptors that account for the charge density (charge per metal atom ratio; q/M) and the size and shape (shape weighted-volume; VS ).


Assuntos
Ânions/química , Cátions/química , Muramidase/química , Compostos de Tungstênio/química , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade
13.
Proc Natl Acad Sci U S A ; 117(8): 4142-4151, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32047034

RESUMO

Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses (D 1/2) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.


Assuntos
Cristalografia por Raios X/métodos , Muramidase/química , Síncrotrons , Temperatura , Cristalização
14.
Int J Mol Sci ; 21(2)2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31941098

RESUMO

Lysozymes are key antimicrobial peptides in the host innate immune system that protect against pathogen infection. In this study, the full-length cDNAs of two c-type lysozymes (gfLyz-C1 and gfLyz-C2) were cloned from goldfish (Carassius auratus). The structural domains, three-dimensional structures, and amino acid sequences of gfLyz-C1 and gfLyz-C2 were highly comparable, as the two proteins shared 89.7% sequence identity. The gfLyz-C1 and gfLyz-C2 recombinant proteins were generated in the insoluble fractions of an Escherichia coli system. Based on the results of lysoplate and turbidimetric assays, gfLyz-C1 and gfLyz-C2 showed broad-spectrum antimicrobial properties with high levels of activity against Micrococcus lysodeikticus, Vibrio parahemolyticus, and Edwardsiella tarda, and relatively low activity against E. coli. Both gfLyz-C1 and gfLyz-C2 mRNAs were mainly expressed in the trunk kidney and head kidney, and gfLyz-C1 was expressed at much higher levels than gfLyz-C2 in the corresponding tissues. The expression of the gfLyz-C1 and gfLyz-C2 transcripts in the trunk kidney and head kidney was induced in these tissues by challenge with heat-inactivated E. coli and lipopolysaccharides (LPS), and the transcriptional responses of gfLyz-C1 were more intense. In goldfish primary trunk kidney cells, the levels of the gfLyz-C1 and gfLyz-C2 transcripts were upregulated by heat-inactivated E. coli, V. parahemolyticus, and E. tarda, as well as LPS, and downregulated by treatment with dexamethasone and leptins. Overall, this study may provide new insights that will improve our understanding of the roles of c-type lysozymes in the innate immunity of cyprinid fish, including the structural and phylogenetic characteristics, antimicrobial effects, and regulatory mechanism.


Assuntos
Anti-Infecciosos , Bactérias/metabolismo , Dexametasona/farmacologia , Proteínas de Peixes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Carpa Dourada , Leptina/farmacologia , Lipopolissacarídeos/toxicidade , Muramidase , Transcrição Genética/efeitos dos fármacos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Proteínas de Peixes/biossíntese , Proteínas de Peixes/química , Proteínas de Peixes/genética , Carpa Dourada/genética , Carpa Dourada/metabolismo , Muramidase/biossíntese , Muramidase/química , Muramidase/genética
15.
Carbohydr Polym ; 231: 115684, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31888826

RESUMO

We developed a rapid and precise method to determine the fraction of acetylation (FA) of unknown chitosan samples using a combination of enzymatic sample hydrolysis, isotopic labeling, and HILIC-ESI-MS analysis. Chitosans are ß-(1,4)-linked, partially N-acetylated and linear polyglucosamines representing an interesting group of functional biopolymers with a broad range of applications. For a better understanding of their structure-function relationships, it is key to have sensitive, accurate structural analysis tools available to determine parameters like the degree of polymerization (DP), fraction of acetylation (FA), or pattern of acetylation (PA). Here, we describe an improved enzymatic/mass spectrometric method for FA analysis of chitosan polymers. In contrast to the original chitinosanase-based mass spectrometric fingerprinting analysis of FA, the new method is independent of the PA and the intermolecular variation in FA (DFA) of the chitosan sample. This allows accurate analysis of heterogeneously de-N-acetylated samples representing the majority of commercially available chitosans.


Assuntos
Biopolímeros/química , Quitina/química , Quitosana/química , Glucosamina/análogos & derivados , Acetilação , Quitina/síntese química , Quitosana/síntese química , Glucosamina/química , Hidrólise , Marcação por Isótopo , Espectrometria de Massas , Muramidase/química
16.
J Agric Food Chem ; 68(7): 2016-2023, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31986021

RESUMO

The protein precipitation (PP) of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by four procyanidin-rich condensed tannin (CT) samples in both 2-[N-morpholino]ethanesulfonic acid (MES) and a modified Goering-Van Soest (GVS) buffer is described. Purified CT samples examined included Vitis vinifera seed (mean degree of polymerization [mDP] 4.1, 16.5% galloylated), Tilia sp. flowers (B-type linkages, mDP 5.9), Vaccinium macrocarpon berries (mDP 8.7, 31.7% A-type linkages). and Trifolium pratense flowers (B-type linkages, mDP 12.3) and were characterized by 2D NMR (>90% purity). In general, CTs precipitated ALF > LYS ≥ BSA. PP in GVS buffer was 1 to 2.25 times greater than that in MES buffer (25 °C). The GVS buffer system better reflects the results/conclusions from the literature on the impacts mDP, galloylation, and A-type linkages have on PP. Determinations of PP using the MES buffer at 37 °C indicated that some of these differences may be attributed to the temperature at which GVS buffer determinations are conducted. In vitro PP studies using the GVS buffer may offer better guidance when selecting CT-containing forages and amendments for ruminant feeding studies.


Assuntos
Biflavonoides/química , Catequina/química , Extratos Vegetais/química , Proteínas de Plantas/química , Proantocianidinas/química , Soroalbumina Bovina/química , Ração Animal/análise , Tampões (Química) , Precipitação Química , Medicago sativa/química , Muramidase/química , Tilia/química , Vaccinium macrocarpon/química , Vitis/química
17.
Mater Sci Eng C Mater Biol Appl ; 108: 110432, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31923974

RESUMO

Coaxial electrospinning with the ability to use simultaneously two separate solvents provides a promising strategy for drug delivery. Nevertheless, controlled release of hydrophilic and sensitive therapeutics from slow biodegradable polymers is still challenging. To address this gap, we fabricated core-sheath fibers for dual delivery of lysozyme, as a model protein, and phenytoin sodium as a small therapeutic molecule. The sheath was processed by a gelatin solution while the core fibers were fabricated from an aqueous gelatin/PVA solution. Microstructural studies by transmission and scanning electron microscopy reveal the formation of homogeneous core-sheath nanofibers with an outer and inner diameter of 180 ± 48 nm and 106 ± 30 nm, respectively. Thermal gravimetric analysis determines that the mass loss of the core-sheath fibers fall between the mass loss values of individual sheath and core fibers. Swelling studies indicate higher water absorption of the core-sheath mat compared to the separate sheath and core membranes. In vitro drug release studies in Phosphate Buffered Saline (PBS) determine sustained release of the therapeutics from the core-sheath structure. The release trails three stages including non-Fickian diffusion at the early stage followed by the Fickian diffusion mechanism. The present study shows a useful approach to design core-sheath nanofibrous membranes with controlled and programmable drug release profiles.


Assuntos
Gelatina , Muramidase , Nanofibras/química , Fenitoína , Álcool de Polivinil , Animais , Linhagem Celular , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Gelatina/química , Gelatina/farmacologia , Camundongos , Muramidase/química , Muramidase/farmacocinética , Muramidase/farmacologia , Fenitoína/química , Fenitoína/farmacocinética , Fenitoína/farmacologia , Álcool de Polivinil/química , Álcool de Polivinil/farmacologia
18.
Soft Matter ; 16(8): 1955-1960, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-31967624

RESUMO

Protein crystals are expected to be useful not only for their molecular structure analysis but also as functional materials due to their unique properties. Although the generation and the propagation of defects during crystallization play critical roles in the final properties of protein crystals, the dynamics of these processes are poorly understood. By time-resolved liquid-cell transmission electron microscopy, we observed that nanosized crystal defects are surprisingly mobile during the early stages of the crystallization of a lysozyme as a model protein. This highly dynamic behavior of defects reveals that the lattice molecules are mobile throughout the crystal structure. Moreover, the disappearance of the defects indicated that intermolecular bonds can break and reform rapidly with little energetic cost, as reported in theoretical studies. All these findings are in marked contrast to the generally accepted notion that crystal lattices are rigid with very limited mobility of individual lattice molecules.


Assuntos
Muramidase/química , Cristalização , Microscopia Eletrônica de Transmissão , Termodinâmica
19.
J Colloid Interface Sci ; 565: 555-566, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31982722

RESUMO

Polyelectrolyte multilayers composed of pharmaceutical grade fucoidan and chitosan have been assembled and studied in terms of their response to physiological solution conditions and the presence of lysozyme. The influence of phosphate buffered saline (PBS) solution on the multilayer was interrogated using attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM). The combination of the techniques reveal that the polyelectrolyte multilayers swell when exposed to PBS after build-up and may include a small degree of mass loss as the film swells. The degree of swelling was influenced by the terminating layer of the multilayer. Upon exposure to lysozyme, it was observed that some deswelling occurred, as the enzyme adsorbed onto and permeated into the multilayer. The behaviour of the multilayer as a potential reservoir for lysozyme contrasts with the interaction with bovine serum albumin, which did not penetrate into the multilayer, indicating either exclusion by size or due to the overall net negative charge of the film.


Assuntos
Quitosana/metabolismo , Muramidase/metabolismo , Polieletrólitos/metabolismo , Polissacarídeos/metabolismo , Quitosana/química , Muramidase/química , Tamanho da Partícula , Polieletrólitos/síntese química , Polieletrólitos/química , Polissacarídeos/química , Propriedades de Superfície
20.
Carbohydr Polym ; 231: 115692, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31888840

RESUMO

Protein-loaded starch microspheres were prepared by water-in-water (w/w) emulsion method. The effects of the molecular weight of starch and protein used, concentration of solutes in both dispersed and continuous phases and starch to protein mass ratio on the yield, loading capacity and encapsulation efficiency were measured. These parameters were significantly higher in Bovine serum albumin (BSA)-loaded microspheres than in lysozyme-loaded microspheres. An increase in the molecular weight of starch, solute concentration in dispersed and continuous phases increased the yield. The encapsulation efficiency was significantly improved when the starch to BSA mass ratio was increased. When the starch to BSA mass ratio was 15:1, the encapsulation efficiency reached about 100 % with a loading capacity of 7.3 g/100 g. This method is more effective when both core (protein) and shell (starch) materials with high molecular weight are used. This approach is environmentally friendly and the processing parameters can be easily optimized.


Assuntos
Microesferas , Muramidase/química , Soroalbumina Bovina/química , Amido/química , Emulsões/química , Peso Molecular , Tamanho da Partícula , Água/química
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