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1.
PLoS One ; 15(7): e0235893, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32692771

RESUMO

Terrestrial nanoplastics (NPs) pose a serious threat to agricultural food production systems due to the potential harm of soil-born micro- and macroorganisms that promote soil fertility and ability of NPs to adsorb onto and penetrate into vegetables and other crops. Very little is known about the dispersion, fate and transport of NPs in soils. This is because of the challenges of analyzing terrestrial NPs by conventional microscopic techniques due to the low concentrations of NPs and absence of optical transparency in these systems. Herein, we investigate the potential utility of small-angle neutron scattering (SANS) and Ultra SANS (USANS) to probe the agglomeration behavior of NPs prepared from polybutyrate adipate terephthalate, a prominent biodegradable plastic used in agricultural mulching, in the presence of vermiculite, an artificial soil. SANS with the contrast matching technique was used to study the aggregation of NPs co-dispersed with vermiculite in aqueous media. We determined the contrast match point for vermiculite was 66% D2O / 33% H2O. At this condition, the signal for vermiculite was ~50-100%-fold lower that obtained using neat H2O or D2O as solvent. According to SANS and USANS, smaller-sized NPs (50 nm) remained dispersed in water and did not undergo size reduction or self-agglomeration, nor formed agglomerates with vermiculite. Larger-sized NPs (300-1000 nm) formed self-agglomerates and agglomerates with vermiculite, demonstrating their significant adhesion with soil. However, employment of convective transport (simulated by ex situ stirring of the slurries prior to SANS and USANS analyses) reduced the self-agglomeration, demonstrating weak NP-NP interactions. Convective transport also led to size reduction of the larger-sized NPs. Therefore, this study demonstrates the potential utility of SANS and USANS with contrast matching technique for investigating behavior of terrestrial NPs in complex soil systems.


Assuntos
Nanoestruturas/análise , Poliésteres/análise , Poluentes do Solo/análise , Solo/química , Nanoestruturas/química , Difração de Nêutrons , Poliésteres/química , Espalhamento a Baixo Ângulo , Poluentes do Solo/química
2.
J Biomed Nanotechnol ; 16(3): 390-397, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32493549

RESUMO

KSL-W peptide has demonstrated antibacterial and antifungal activity and inhibitory effects against oral biofilm. This study aimed to check out the effect of chlorhexidine (CLX) or KSL-W peptide-loaded poloxamer 407-based microemulsions for buccal delivery on Fusobacterium nucleatum (F. nucleatum) biofilm. The formulation (F) containing 10% copolymer poloxamer 407 dispersion (1%), 40% oleic acid and 50% PPG-5-CETETH-20 was characterized by polarized light microscopy (PLM), small-angle X-ray scattering (SAXS), rheology, bioadhesive and syringeability; and in the treatment of a biofilm produced by F. nucleatum. The darkfield images obtained by PLM and the SAXS curves with an extended peak indicated that the system was characteristic of microemulsions. In a continuous analysis, microemulsions exhibited Newtonian behavior. In frequency, the oscillatory analysis profile presented predominantly viscous behavior. Bioadhesive force detected in the analysis of F (7.4 ± 1.81 mN˙ s) and syringeability (17.83± 5.97 N · mm) being adequate values for buccal administration. After 4 h, KSL-W-loaded F shown over 20% higher effectiveness than chlorhexidine-loaded microemulsions. In conclusion, the KSL-W-loaded microemulsions showed a considerable reduction in F. nucleatum biofilm formation and presented promising structural properties for buccal drug delivery.


Assuntos
Fusobacterium nucleatum , Peptídeos Catiônicos Antimicrobianos , Biofilmes , Poloxâmero , Espalhamento a Baixo Ângulo , Difração de Raios X
3.
Nucleic Acids Res ; 48(14): 8006-8021, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32556302

RESUMO

The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.


Assuntos
Iniciação Traducional da Cadeia Peptídica , Poliovirus/genética , RNA Viral/química , Proteínas de Ligação a RNA/química , Microscopia Crioeletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
4.
Biochem Biophys Res Commun ; 527(3): 618-623, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: covidwho-155056

RESUMO

The nucleocapsid (N) protein is an important antigen for coronavirus, which participate in RNA package and virus particle release. In this study, we expressed the N protein of SARS-CoV-2 and characterized its biochemical properties. Static light scattering, size exclusive chromatography, and small-angle X-ray scattering (SAXS) showed that the purified N protein is largely a dimer in solution. CD spectra showed that it has a high percentage of disordered region at room temperature while it was best structured at 55 °C, suggesting its structural dynamics. Fluorescence polarization assay showed it has non-specific nucleic acid binding capability, which raised a concern in using it as a diagnostic marker. Immunoblot assays confirmed the presence of IgA, IgM and IgG antibodies against N antigen in COVID-19 infection patients' sera, proving the importance of this antigen in host immunity and diagnostics.


Assuntos
Betacoronavirus/química , Proteínas do Nucleocapsídeo/química , Anticorpos Antivirais/sangue , Infecções por Coronavirus , Humanos , Ácidos Nucleicos , Pandemias , Pneumonia Viral , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X
5.
Nat Commun ; 11(1): 2495, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427872

RESUMO

Colloidal crystal engineering with nucleic acid-modified nanoparticles is a powerful way for preparing 3D superlattices, which may be useful in many areas, including catalysis, sensing, and photonics. To date, the building blocks studied have been primarily based upon metals, metal oxides, chalcogenide semiconductors, and proteins. Here, we show that metal-organic framework nanoparticles (MOF NPs) densely functionalized with oligonucleotides can be programmed to crystallize into a diverse set of superlattices with well-defined crystal symmetries and compositions. Electron microscopy and small-angle X-ray scattering characterization confirm the formation of single-component MOF superlattices, binary MOF-Au single crystals, and two-dimensional MOF nanorod assemblies. Importantly, DNA-modified porphyrinic MOF nanorods (PCN-222) were assembled into 2D superlattices and found to be catalytically active for the photooxidation of 2-chloroethyl ethyl sulfide (CEES, a chemical warfare simulant of mustard gas). Taken together, these new materials and methods provide access to colloidal crystals that incorporate particles with the well-established designer properties of MOFs and, therefore, increase the scope of possibilities for colloidal crystal engineering with DNA.


Assuntos
Coloides/química , DNA/química , Estruturas Metalorgânicas/química , Nanopartículas/química , Cristalização , DNA/genética , Engenharia/métodos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Nanopartículas/ultraestrutura , Nanotubos/química , Nanotubos/ultraestrutura , Tamanho da Partícula , Espalhamento a Baixo Ângulo , Prata/química , Difração de Raios X
6.
Food Chem ; 327: 127000, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32454284

RESUMO

Iron oxides used as food colorants are listed in the European Union with the number E172. However, there are no specifications concerning the fraction of nanoparticles in these pigments. Here, seven E172 products were thoroughly characterized. Samples of all colors were analyzed with a broad spectrum of methods to assess their physico-chemical properties. Small-Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), zeta-potential, Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), X-ray diffraction (XRD), Brunauer-Emmett-Teller analysis (BET), Asymmetric Flow Field-Flow Fractionation (AF4) and in vitro cell viability measurements were used. Nanoparticles were detected in all E172 samples by TEM or SAXS measurements. Quantitative results from both methods were comparable. Five pigments were evaluated by TEM, of which four had a size median below 100 nm, while SAXS showed a size median below 100 nm for six evaluated pigments. Therefore, consumers may be exposed to iron oxide nanoparticles through the consumption of food pigments.


Assuntos
Compostos Férricos/química , Corantes de Alimentos/química , Difusão Dinâmica da Luz , Fracionamento por Campo e Fluxo/métodos , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Tamanho da Partícula , Espalhamento a Baixo Ângulo , Difração de Raios X
7.
Biochem Biophys Res Commun ; 527(3): 618-623, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32416961

RESUMO

The nucleocapsid (N) protein is an important antigen for coronavirus, which participate in RNA package and virus particle release. In this study, we expressed the N protein of SARS-CoV-2 and characterized its biochemical properties. Static light scattering, size exclusive chromatography, and small-angle X-ray scattering (SAXS) showed that the purified N protein is largely a dimer in solution. CD spectra showed that it has a high percentage of disordered region at room temperature while it was best structured at 55 °C, suggesting its structural dynamics. Fluorescence polarization assay showed it has non-specific nucleic acid binding capability, which raised a concern in using it as a diagnostic marker. Immunoblot assays confirmed the presence of IgA, IgM and IgG antibodies against N antigen in COVID-19 infection patients' sera, proving the importance of this antigen in host immunity and diagnostics.


Assuntos
Betacoronavirus/química , Proteínas do Nucleocapsídeo/química , Anticorpos Antivirais/sangue , Infecções por Coronavirus , Humanos , Ácidos Nucleicos , Pandemias , Pneumonia Viral , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X
8.
PLoS One ; 15(5): e0233131, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392265

RESUMO

Human skin, not previously frozen, was studied by small-angle X-ray diffraction. The samples were folded so that a 6µm X-ray beam passed through the top layer of skin, stratum corneum. Diffraction patterns recorded with this method consisted of peaks at about q = 0.5, 1.0 and 1.4 nm-1 in the direction perpendicular to the skin surface more clearly than in previous studies. These peaks are interpreted to arise from lipids between corneocytes. A simple unit of a linear electron density profile with three minima was used to account for the observed intensity profiles. Combinations of calculated diffraction from models with one, two and three units accounted for the major part of the observed diffraction pattern, showing the diversity in the structure of the intercellular lipids.


Assuntos
Epiderme/química , Lipídeos/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Adulto , Feminino , Humanos , Pessoa de Meia-Idade
9.
Food Chem ; 321: 126717, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32259734

RESUMO

Effectiveness of liposomes elaborated with rapeseed phospholipid (RP) extracted from a residue of oil processing, stigmasterol (ST) and/or hydrogenated phosphatidylcholine (HPC) for the encapsulation lactoferrin (LF) was studied; lipid membrane of liposomes was characterized (bilayer size, chain conformational order, lateral packing, lipid phase, and morphology) and the protection offered to the encapsulated LF during in vitro digestion was determined. Liposomes composed of RP+STLC(low concentration) showed spherical and irregular vesicles without perforations. Lamellar structure was organized in a liquid-ordered phase with a potential orthorhombic packing. Stability and size of the liposomes were more affected by gastric digestion than intestinal digestion; 67-80% of the initially encapsulated LF remained intact after gastric digestion whereas the percentage was reduced to 16-35% after intestinal digestion. Our results shows that liposomes elaborated with RP, properly combined with other lipids, can be a useful oral delivery system of molecules sensitive to digestive enzymes.


Assuntos
Lactoferrina/farmacocinética , Lipossomos/química , Fosfolipídeos/química , Administração Oral , Brassica napus/química , Digestão , Suco Gástrico/metabolismo , Absorção Intestinal , Lactoferrina/química , Lipossomos/farmacocinética , Fosfatidilcolinas/química , Espalhamento a Baixo Ângulo , Difração de Raios X
10.
PLoS Comput Biol ; 16(4): e1007870, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339173

RESUMO

Many proteins contain multiple folded domains separated by flexible linkers, and the ability to describe the structure and conformational heterogeneity of such flexible systems pushes the limits of structural biology. Using the three-domain protein TIA-1 as an example, we here combine coarse-grained molecular dynamics simulations with previously measured small-angle scattering data to study the conformation of TIA-1 in solution. We show that while the coarse-grained potential (Martini) in itself leads to too compact conformations, increasing the strength of protein-water interactions results in ensembles that are in very good agreement with experiments. We show how these ensembles can be refined further using a Bayesian/Maximum Entropy approach, and examine the robustness to errors in the energy function. In particular we find that as long as the initial simulation is relatively good, reweighting against experiments is very robust. We also study the relative information in X-ray and neutron scattering experiments and find that refining against the SAXS experiments leads to improvement in the SANS data. Our results suggest a general strategy for studying the conformation of multi-domain proteins in solution that combines coarse-grained simulations with small-angle X-ray scattering data that are generally most easy to obtain. These results may in turn be used to design further small-angle neutron scattering experiments that exploit contrast variation through 1H/2H isotope substitutions.


Assuntos
Simulação de Dinâmica Molecular , Proteínas , Espalhamento a Baixo Ângulo , Difração de Raios X , Algoritmos , Biologia Computacional , Nêutrons , Conformação Proteica , Domínios Proteicos , Proteínas/análise , Proteínas/química
11.
Proc Natl Acad Sci U S A ; 117(16): 8870-8875, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32245816

RESUMO

The ability to precisely design large proteins with diverse shapes would enable applications ranging from the design of protein binders that wrap around their target to the positioning of multiple functional sites in specified orientations. We describe a protein backbone design method for generating a wide range of rigid fusions between helix-containing proteins and use it to design 75,000 structurally unique junctions between monomeric and homo-oligomeric de novo designed and ankyrin repeat proteins (RPs). Of the junction designs that were experimentally characterized, 82% have circular dichroism and solution small-angle X-ray scattering profiles consistent with the design models and are stable at 95 °C. Crystal structures of four designed junctions were in close agreement with the design models with rmsds ranging from 0.9 to 1.6 Å. Electron microscopic images of extended tetrameric structures and ∼10-nm-diameter "L" and "V" shapes generated using the junctions are close to the design models, demonstrating the control the rigid junctions provide for protein shape sculpting over multiple nanometer length scales.


Assuntos
Modelos Moleculares , Engenharia de Proteínas/métodos , Proteínas/ultraestrutura , Sequências Repetitivas de Aminoácidos/genética , Dicroísmo Circular , Microscopia Eletrônica , Biblioteca de Peptídeos , Conformação Proteica em alfa-Hélice/genética , Dobramento de Proteína , Proteínas/química , Proteínas/genética , Espalhamento a Baixo Ângulo , Difração de Raios X
12.
Biochim Biophys Acta Biomembr ; 1862(6): 183256, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32145283

RESUMO

Living matter is a quasi-stationary out-of-equilibrium system; in this physical condition, structural fluctuations at nano- and meso-scales are needed to understand the physics behind its biological functionality. Myelin has a simple ultrastructure whose fluctuations show correlated disorder in its functional out-of-equilibrium state. However, there is no information on the relationship between this correlated disorder and the dynamics of the intrinsically disordered Myelin Basic Protein (MBP) which is expected to influence the membrane structure and overall functionality. In this work, we have investigated the role of this protein structural dynamics in the myelin ultrastructure fluctuations in various conditions, by using synchrotron Scanning micro X Ray Diffraction and Small Angle X ray Scattering. We have induced the crossover from out-of-equilibrium functional state to in-equilibrium degeneration changing the pH to values far from physiological condition. The observed compression of the cytosolic layer thickness probes that the intrinsic large MBP fluctuations preserve the cytosol structure also in the degraded state. Thus, the transition of myelin ultrastructure from correlated to uncorrelated disordered state, is principally affected by the deformation of the membrane and extracellular domain.


Assuntos
Simulação de Dinâmica Molecular , Proteína Básica da Mielina/química , Bainha de Mielina/ultraestrutura , Animais , Estruturas da Membrana Celular , Humanos , Proteínas Intrinsicamente Desordenadas , Bainha de Mielina/química , Proteólise , Espalhamento a Baixo Ângulo , Síncrotrons , Difração de Raios X
13.
Phys Chem Chem Phys ; 22(13): 6984-6992, 2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-32188961

RESUMO

Ectoine is a small zwitterionic osmolyte and compatible solute, which does not interfere with cell metabolism even at molar concentrations. Plasmid DNA (pUC19) was irradiated with ultraviolet radiation (UV-C at 266 nm) under quasi physiological conditions (PBS) and in pure water in the presence and absence of ectoine (THP(B)) and hydroxyectoine (THP(A)). Different types of UV induced DNA damage were analysed: DNA single-strand breaks (SSBs), abasic sites and cyclobutane pyrimidine dimers (CPDs). A complex interplay between these factors was observed with respect to the nature and occurrence of DNA damage with 266 nm photons. In PBS, the cosolutes showed efficient protection against base damage, whilst in pure water, a dramatic shift from SSB damage to base damage was observed when cosolutes were added. To test whether these effects are caused by ectoine binding to DNA, further experiments were conducted: small-angle X-ray scattering (SAXS), surface-plasmon resonance (SPR) measurements and Raman spectroscopy. The results show, for the first time, a close interaction between ectoine and DNA. This is in stark contrast to the assumption made by preferential exclusion models, which are often used to interpret the behaviour of compatible solutes within cells and with biomolecules. It is tentatively proposed that the alterations of UV damage to DNA are attributed to ectoine influence on nucleobases through the direct interaction between ectoine and DNA.


Assuntos
Diamino Aminoácidos/metabolismo , Dano ao DNA/genética , DNA/metabolismo , DNA/efeitos da radiação , Raios Ultravioleta , DNA/química , Plasmídeos/química , Plasmídeos/metabolismo , Plasmídeos/efeitos da radiação , Espalhamento a Baixo Ângulo , Ressonância de Plasmônio de Superfície , Difração de Raios X
14.
Food Chem ; 317: 126464, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32114275

RESUMO

Oat rice kernels were subjected to decortication (DOR), decortication and enzyme deactivation (DDOR), decortication and cooking (DCOR), as well as combined decortication, enzyme deactivation and cooking (DDCOR). The starch fractions were isolated and their structural features were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, solid-state 13C nuclear magnetic resonance, small angle X-ray scattering (SAXS), and scanning electron microscope. In the cooked oat rice samples (DCOR and DDCOR), in addition to losing a significant amount of the A-type crystalline structure, there was an enhancement in the proportion of V-type crystallinity. The cooking process completely destroyed the periodic lamellar structure of oat starch on the SAXS profile. The Mw values (1.195 × 107-1.459 × 107 g/mol) were in the following order: DOR > DDOR > DCOR > DDCOR. The data was in line with the results for crystallinity, double helix content, degree of order, melting enthalpy, and those obtained for textural parameters, resistant starch content, and bile acid binding capacity.


Assuntos
Avena/química , Culinária/métodos , Amido/química , Cristalização , Enzimas/química , Espectroscopia de Ressonância Magnética , Espalhamento a Baixo Ângulo , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
15.
Food Chem ; 318: 126487, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32126469

RESUMO

This study aimed at exploring the effects of exogenous seed crystals on the starch recrystallization. A-type and B-type seed crystals from rice starch were added to rice starch paste during cooling to analyze the changes in retrogradation process. DSC results indicated that B-type seed improved the uniformity of crystal size and the degree of crystal perfection; it also affected the nucleation mode of retrograded starches at 4 °C, while A-type seed did not affect these properties. The XRD showed that the unit cell was compact, and the crystal grain became larger upon the introduction of B-type seed, while A-type seemed to loosen the cell. The SAXS reflected that B-type seed increased the thickness of the imperfect lamellar structure formed during recrystallization, while A-type seed decreased it. These findings might be due to the molecular segment arrangement on the nuclei growth surface affecting the arrangement of starch molecular segments approaching the nuclei.


Assuntos
Oryza/química , Amido/química , Cristalização , Espalhamento a Baixo Ângulo , Sementes/química , Difração de Raios X
16.
Biochemistry ; 59(12): 1252-1260, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32176479

RESUMO

Protein kinase R (PKR) is a key antiviral component of the innate immune pathway and is activated by viral double-stranded RNAs (dsRNAs). Adenovirus-associated RNA 1 (VAI) is an abundant, noncoding viral RNA that functions as a decoy by binding PKR but not inducing activation, thereby inhibiting the antiviral response. In VAI, coaxial stacking produces an extended helix that mediates high-affinity PKR binding but is too short to result in activation. Like adenovirus, Epstein-Barr virus produces high concentrations of a noncoding RNA, EBER1. Here, we compare interactions of PKR with VAI and EBER1 and present a structural model of EBER1. Both RNAs function as inhibitors of dsRNA-mediated PKR activation. However, EBER1 weakly activates PKR whereas VAI does not. PKR binds EBER1 more weakly than VAI. Assays at physiological ion concentrations indicate that both RNAs can accommodate two PKR monomers and induce PKR dimerization. A structural model of EBER1 was obtained using constraints derived from chemical structure probing and small-angle X-ray scattering experiments. The central stem of EBER1 coaxially stacks with stem loop 4 and stem loop 1 to form an extended RNA duplex of ∼32 bp that binds PKR and promotes activation. Our observations that EBER1 binds PKR much more weakly than VAI and exhibits weak PKR activation suggest that EBER1 is less well suited to function as an RNA decoy.


Assuntos
Herpesvirus Humano 4/genética , Interações entre Hospedeiro e Microrganismos/genética , RNA Viral/metabolismo , eIF-2 Quinase/genética , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/imunologia , Humanos , Imunidade Inata/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Multimerização Proteica/genética , Multimerização Proteica/imunologia , Estabilidade de RNA , RNA Viral/química , Espalhamento a Baixo Ângulo , Difração de Raios X , eIF-2 Quinase/química , eIF-2 Quinase/imunologia , eIF-2 Quinase/metabolismo
17.
Nucleic Acids Res ; 48(10): 5720-5734, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32083666

RESUMO

The structure of the 68 nt sequence with G-quadruplex forming potential within the hTERT promoter is disputed. One model features a structure with three stacked parallel G-quadruplex units, while another features an unusual duplex hairpin structure adjoined to two stacked parallel and antiparallel quadruplexes. We report here the results of an integrated structural biology study designed to distinguish between these possibilities. As part of our study, we designed a sequence with an optimized hairpin structure and show that its biophysical and biochemical properties are inconsistent with the structure formed by the hTERT wild-type sequence. By using circular dichroism, thermal denaturation, nuclear magnetic resonance spectroscopy, analytical ultracentrifugation, small-angle X-ray scattering, molecular dynamics simulations and a DNase I cleavage assay we found that the wild type hTERT core promoter folds into a stacked, three-parallel G-quadruplex structure. The hairpin structure is inconsistent with all of our experimental data obtained with the wild-type sequence. All-atom models for both structures were constructed using molecular dynamics simulations. These models accurately predicted the experimental hydrodynamic properties measured for each structure. We found with certainty that the wild-type hTERT promoter sequence does not form a hairpin structure in solution, but rather folds into a compact stacked three-G-quadruplex conformation.


Assuntos
Quadruplex G , Regiões Promotoras Genéticas , Telomerase/genética , Sequência de Bases , Dicroísmo Circular , DNA/química , Humanos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Desnaturação de Ácido Nucleico , Espalhamento a Baixo Ângulo , Difração de Raios X
18.
Food Chem ; 315: 126245, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32004980

RESUMO

Our objective was to investigate the correlation between processing conditions and structural properties of lotus seed starch-lecithin complexes by dynamic high-pressure homogenization and explore the formation mechanism. The complexes formed with 5% lecithin at 90 MPa had the highest complex index among samples, thus protecting the integrity of the particles. The complexes inhibited the degradation of amylopectin and retrogradation of amylose, and displayed different V6II-, V6I- and A-type crystalline patterns. Additionally, the double helix structure was enhanced with increasing pressure, and the addition of lecithin contributed to the formation of single-helix amylose-lecithin complexes. These complexes prevented the single helix structure of starch to further form double helix structures, as demonstrated by visual correlation analysis. Moreover, a formation mechanism was established, and lotus seed starch-lecithin complexes with V6I-type crystalline were formed under appropriate conditions, but a homogenization pressure either too low or too high was not conducive to complex formation.


Assuntos
Manipulação de Alimentos/métodos , Lecitinas/química , Lotus/química , Amido/química , Amilopectina/química , Amilose/química , Cromatografia em Gel , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura , Pressão , Espalhamento a Baixo Ângulo , Sementes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
19.
Nat Commun ; 11(1): 1026, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32094331

RESUMO

Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an α/ß-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.


Assuntos
Domínio Catalítico , Esterases/metabolismo , Proteínas Fúngicas/metabolismo , Ácido Glucurônico/metabolismo , Polyporales/enzimologia , Carboidratos , Parede Celular/metabolismo , Cristalografia por Raios X , Esterases/isolamento & purificação , Esterases/ultraestrutura , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Hidrólise , Lignina/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Especificidade por Substrato , Difração de Raios X
20.
Mol Pharmacol ; 97(4): 295-303, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32102968

RESUMO

The main objective of this study was to clarify the topical mechanisms underlying diclofenac-induced gastric toxicity by considering for the first time both ionization states of this nonsteroidal anti-inflammatory drug. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes were the model system chosen to mimic the protective phospholipid layers of the gastric mucosa and to describe the interactions with diclofenac, considering the pH gradient found in the gastric mucosa (3 < pH < 7.4). Complementary experimental techniques were combined to evaluate the drug's affinity for DMPC bilayers, as well as to assess the drug's effects on the structural properties of the phospholipid bilayer. The diclofenac-DMPC interactions were clearly dependent on the drug's ionization state. Neutral diclofenac displayed greater affinity for DMPC bilayers than anionic diclofenac. Moreover, the protonated/neutral form of the drug induced more pronounced and/or distinct alterations in the structure of the DMPC bilayer than the deprotonated/ionized form, considering similar membrane concentrations. Therefore, neutral diclofenac-induced changes in the structural properties of the external phospholipid layers of the gastric mucosa may constitute an additional toxicity mechanism of this worldwide-used drug, which shall be considered for the development of safer therapeutic strategies. SIGNIFICANCE STATEMENT: Neutral or anionic diclofenac exerted distinct alterations in phosphatidylcholine bilayers, which are used in this work as models for the protective phospholipid layers of the gastric mucosa. Remarkable changes were induced by neutral diclofenac in the structural properties of the phospholipid bilayer, suggesting that both ionized and neutral states of nonsteroidal anti-inflammatory drugs must be considered to clarify their mechanisms of toxicity and to ultimately develop safer anti-inflammatory drugs.


Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Diclofenaco/toxicidade , Dimiristoilfosfatidilcolina/química , Mucosa Gástrica/efeitos dos fármacos , Bicamadas Lipídicas/química , Mucosa Gástrica/química , Concentração de Íons de Hidrogênio , Lipossomos/química , Estrutura Molecular , Espalhamento a Baixo Ângulo , Difração de Raios X
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