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1.
Nat Commun ; 11(1): 4372, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873801

RESUMO

3D molecular structure determination is a challenge for organic compounds or natural products available in minute amounts. Proton/proton and proton/carbon correlations yield the constitution. J couplings and NOEs oftentimes supported by one-bond 1H,13C residual dipolar couplings (RDCs) or by 13C residual chemical shift anisotropies (RCSAs) provide the relative configuration. However, these RDCs or carbon RCSAs rely on 1% natural abundance of 13C preventing their use for compounds available only in quantities of a few 10's of µgs. By contrast, 1H RCSAs provide similar information on spatial orientation of structural moieties within a molecule, while using the abundant 1H spin. Herein, 1H RCSAs are accurately measured using constrained aligning gels or liquid crystals and applied to the 3D structural determination of molecules with varying complexities. Even more, deuterated alignment media allow the elucidation of the relative configuration of around 35 µg of a briarane compound isolated from Briareum asbestinum.


Assuntos
Antozoários/química , Produtos Biológicos/química , Diterpenos/química , Conformação Molecular , Prótons , Animais , Anisotropia , Espectroscopia de Prótons por Ressonância Magnética
2.
J Phys Chem Lett ; 11(19): 8084-8093, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32885971

RESUMO

SARS-CoV-2 is a health threat with dire socioeconomical consequences. As the crucial mediator of infection, the viral glycosylated spike protein (S) has attracted the most attention and is at the center of efforts to develop therapeutics and diagnostics. Herein, we use an original decomposition approach to identify energetically uncoupled substructures as antibody binding sites on the fully glycosylated S. Crucially, all that is required are unbiased MD simulations; no prior knowledge of binding properties or ad hoc parameter combinations is needed. Our results are validated by experimentally confirmed structures of S in complex with anti- or nanobodies. We identify poorly coupled subdomains that are poised to host (several) epitopes and potentially involved in large functional conformational transitions. Moreover, we detect two distinct behaviors for glycans: those with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes, and those with weaker coupling could themselves be prone to antibody recognition.


Assuntos
Epitopos/química , Simulação de Dinâmica Molecular , Glicoproteína da Espícula de Coronavírus/química , Algoritmos , Betacoronavirus/química , Sítios de Ligação de Anticorpos , Glicosilação , Humanos , Modelos Moleculares , Conformação Molecular , Peptídeos/química , Polissacarídeos/química
3.
Nat Commun ; 11(1): 4747, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958761

RESUMO

Chromosome structure at the multi-nucleosomal level has remained ambiguous in spite of its central role in epigenetic regulation and genome dynamics. Recent investigations of chromatin architecture portray diverse modes of interaction within and between nucleosome chains, but how this is realized at the atomic level is unclear. Here we present near-atomic resolution crystal structures of nucleosome fibres that assemble from cohesive-ended dinucleosomes with and without linker histone. As opposed to adopting folded helical '30 nm' structures, the fibres instead assume open zigzag conformations that are interdigitated with one another. Zigzag conformations obviate extreme bending of the linker DNA, while linker DNA size (nucleosome repeat length) dictates fibre configuration and thus fibre-fibre packing, which is supported by variable linker histone binding. This suggests that nucleosome chains have a predisposition to interdigitate with specific characteristics under condensing conditions, which rationalizes observations of local chromosome architecture and the general heterogeneity of chromatin structure.


Assuntos
Nucleossomos/química , Nucleossomos/metabolismo , Sequência de Bases , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica
4.
Nature ; 585(7824): 303-308, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879488

RESUMO

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.


Assuntos
Anestésicos Gerais/química , Anestésicos Gerais/farmacologia , Barbitúricos/química , Barbitúricos/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Microscopia Crioeletrônica , Receptores de GABA-A/química , Regulação Alostérica/efeitos dos fármacos , Anestésicos Gerais/metabolismo , Barbitúricos/metabolismo , Benzodiazepinas/metabolismo , Bicuculina/química , Bicuculina/metabolismo , Bicuculina/farmacologia , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Diazepam/química , Diazepam/metabolismo , Diazepam/farmacologia , Eletrofisiologia , Etomidato/química , Etomidato/metabolismo , Etomidato/farmacologia , Flumazenil/farmacologia , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/metabolismo , Antagonistas de Receptores de GABA-A/farmacologia , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Fenobarbital/química , Fenobarbital/metabolismo , Fenobarbital/farmacologia , Picrotoxina/química , Picrotoxina/metabolismo , Picrotoxina/farmacologia , Propofol/química , Propofol/metabolismo , Propofol/farmacologia , Receptores de GABA-A/metabolismo , Receptores de GABA-A/ultraestrutura , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologia
5.
Nat Commun ; 11(1): 4734, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948759

RESUMO

A primary reason for the intense interest in structural biology is the fact that knowledge of structure can elucidate macromolecular functions in living organisms. Sustained effort has resulted in an impressive arsenal of tools for determining the static structures. But under physiological conditions, macromolecules undergo continuous conformational changes, a subset of which are functionally important. Techniques for capturing the continuous conformational changes underlying function are essential for further progress. Here, we present chemically-detailed conformational movies of biological function, extracted data-analytically from experimental single-particle cryo-electron microscopy (cryo-EM) snapshots of ryanodine receptor type 1 (RyR1), a calcium-activated calcium channel engaged in the binding of ligands. The functional motions differ substantially from those inferred from static structures in the nature of conformationally active structural domains, the sequence and extent of conformational motions, and the way allosteric signals are transduced within and between domains. Our approach highlights the importance of combining experiment, advanced data analysis, and molecular simulations.


Assuntos
Agonistas dos Canais de Cálcio/química , Substâncias Macromoleculares/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Sítios de Ligação , Microscopia Crioeletrônica , Ligantes , Conformação Molecular , Simulação de Dinâmica Molecular , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
6.
J Chromatogr A ; 1628: 461448, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822987

RESUMO

During a screening of cyclodextrins (CDs) as chiral selectors for the separation of daclatasvir (DCV) and its enantiomer by capillary electrophoresis (CE), an unusual phenomenon for CDs was observed, that is two peaks with a plateau in between using γ-CD as chiral selector. The same result was encountered when enantiopure DCV was injected or when analyzing a sample containing enantiopure DCV and γ-CD in a CD-free background electrolyte. Peak coalescence was observed at 45°C and at a pH above 3.5. Two peaks with a plateau were also observed for DCV stereoisomers as well as a structural analog. However, only a single peak was detected if one or both amino acid moieties of DCV were lacking. Nuclear magnetic resonance (NMR) experiments including Nuclear Overhauser effect-based methods showed that in solution DCV adopted a folded conformation in which the isopropyl side chain of the valine residues pointed toward the aromatic rings of DCV. Moreover, NMR unequivocally demonstrated the simultaneous formation of DCV-γ-CD inclusion complexes with 1:1 and 2:1 stoichiometry, which was corroborated by mass spectrometry. In both complexes, DCV also adopted a folded structure. The RSSR-diastereomer of DCV as well as an analog lacking one of the amino acid moieties also formed 1:1 and 2:1 complexes with γ-CD although a plateau was only observed in the case of the RSSR-diastereomer. As shown by CE-MS, both DCV-γ-CD complexes surprisingly comigrated as the first peak, while the second migrating peak represents non-complexed DCV.


Assuntos
Imidazóis/química , gama-Ciclodextrinas/química , Eletroforese Capilar , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Conformação Molecular , Estereoisomerismo
7.
Acta Crystallogr C Struct Chem ; 76(Pt 8): 795-809, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32756043

RESUMO

The crystal structures of four new chiral [1,2,3]triazolo[5,1-b][1,3,4]thiadiazines are described, namely, ethyl 5'-benzoyl-5'H,7'H-spiro[cyclohexane-1,6'-[1,2,3]triazolo[5,1-b][1,3,4]thiadiazine]-3'-carboxylate, C19H22N4O3S, ethyl 5'-(4-methoxybenzoyl)-5'H,7'H-spiro[cyclohexane-1,6'-[1,2,3]triazolo[5,1-b][1,3,4]thiadiazine]-3'-carboxylate, C20H24N4O4S, ethyl 6,6-dimethyl-5-(4-methylbenzoyl)-6,7-dihydro-5H-[1,2,3]triazolo[5,1-b][1,3,4]thiadiazine-3-carboxylate, C17H20N4O3S, and ethyl 5-benzoyl-6-(4-methoxyphenyl)-6,7-dihydro-5H-[1,2,3]triazolo[5,1-b][1,3,4]thiadiazine-3-carboxylate, C21H20N4O4S. The crystallographic data and cell activities of these four compounds and of the structures of three previously reported similar compounds, namely, ethyl 5'-(4-methylbenzoyl)-5'H,7'H-spiro[cyclopentane-1,6'-[1,2,3]triazolo[5,1-b][1,3,4]thiadiazine]-3'-carboxylate, C19H22N4O3S, ethyl 5'-(4-methoxybenzoyl)-5'H,7'H-spiro[cyclopentane-1,6'-[1,2,3]triazolo[5,1-b][1,3,4]thiadiazine]-3'-carboxylate, C19H22N4O4S, and ethyl 6-methyl-5-(4-methylbenzoyl)-6-phenyl-6,7-dihydro-5H-[1,2,3]triazolo[5,1-b][1,3,4]thiadiazine-3-carboxylate, C22H22N4O3S, are contrasted and compared. For both crystallization and an MTT assay, racemic mixtures of the corresponding [1,2,3]triazolo[5,1-b][1,3,4]thiadiazines were used. The main manner of molecular packing in these compounds is the organization of either enantiomeric pairs or dimers. In both cases, the formation of two three-centre hydrogen bonds can be detected resulting from intramolecular N-H...O and intermolecular N-H...O or N-H...N interactions. Molecules of different enantiomeric forms can also form chains through N-H...O hydrogen bonds or form layers between which only weak hydrophobic contacts exist. Unlike other [1,2,3]triazolo[5,1-b][1,3,4]thiadiazines, ethyl 5'-benzoyl-5'H,7'H-spiro[cyclohexane-1,6'-[1,2,3]triazolo[5,1-b][1,3,4]thiadiazine]-3'-carboxylate contains molecules of only the (R)-enantiomer; moreover, the N-H group does not participate in any significant intermolecular interactions. Molecular mechanics methods (force field OPLS3e) and the DFT B3LYP/6-31G+(d,p) method show that the compound forming enantiomeric pairs via weak N-H...N hydrogen bonds is subject to greater distortion of the geometry under the influence of the intermolecular interactions in the crystal. For intramolecular N-H...O and S...O interactions, an analysis of the noncovalent interactions (NCIs) was carried out. The cellular activities of the compounds were tested by evaluating their antiproliferative effect against two normal human cell lines and two cancer cell lines in terms of half-maximum inhibitory concentration (IC50). Some derivatives have been found to be very effective in inhibiting the growth of Hela cells at nanomolar and submicromolar concentrations with minimal cytotoxicity in relation to normal cells.


Assuntos
Cicloexanos/química , Compostos Heterocíclicos/farmacologia , Tiadiazinas/química , Cristalografia por Raios X , Células HeLa , Compostos Heterocíclicos/química , Humanos , Ligação de Hidrogênio , Conformação Molecular , Estereoisomerismo , Tiadiazinas/farmacologia
8.
Nat Protoc ; 15(9): 2980-3008, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32839575

RESUMO

High-surface-area mesoporous materials expose abundant functional sites for improved performance in applications such as gas storage/separation, catalysis, and sensing. Recently, soft templates composed of amphiphilic surfactants and block copolymers have been used to introduce mesoporosity in various materials, including metals, metal oxides and carbonaceous compounds. In particular, mesoporous metals are attractive in electrocatalysis because their porous networks expose numerous unsaturated atoms on high-index facets that are highly active in catalysis. In this protocol, we describe how to create mesoporous metal films composed of gold, palladium, or platinum using block copolymer micelle templates. The amphiphilic block copolymer micelles are the sacrificial templates and generate uniform structures with tunable pore sizes in electrodeposited metal films. The procedure describes the electrodeposition in detail, including parameters such as micelle diameters, deposition potentials, and deposition times to ensure reproducibility. The micelle diameters can be controlled by swelling the micelles with different solvent mixtures or by using block copolymer micelles with different molecular weights. The deposition potentials and deposition times allow further control of the mesoporous structure and its thickness, respectively. Procedures for example applications are included: glucose oxidation, ethanol oxidation and methanol oxidation reactions. The synthetic methods for preparation of mesoporous metal films will take ~4 h; the subsequent electrochemical tests will take ~5 h for glucose sensing and ~3 h for alcohol oxidation reaction.


Assuntos
Ouro/química , Paládio/química , Platina/química , Álcoois/química , Catálise , Técnicas de Química Sintética , Eletroquímica , Interações Hidrofóbicas e Hidrofílicas , Micelas , Modelos Moleculares , Conformação Molecular , Oxirredução , Polímeros/química
9.
J Oleo Sci ; 69(9): 1125-1132, 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32788521

RESUMO

Controlling the size of nanoparticles is important for drug delivery methods such as pulmonary administration, transdermal administration, and intravenous administration. In this study, we have investigated the effect of polymer conformation in organic solvents on the size of the nanoparticles. Poly(L-lactide-co-glycolide) (PLLGA), a promising nanoparticle carrier, was used as the polymer. A mixed solution of dichloromethane, which is a good solvent, and a lower alcohol (methanol, ethanol, and 1-propanol), which is a poor solvent, was used as the solvent for dissolving PLLGA. An oil-in-water emulsion was prepared by sonication using the mixed solution of organic solvents in which PLLGA was dissolved as a dispersed phase and an amino acid aqueous solution as a continuous phase. Nanocomposite particles were prepared from the emulsion using a spray dryer and redispersed in purified water to obtain the PLLGA nanoparticles. The conformation of PLLGA molecules in the organic solvents was evaluated by analyzing the results of the viscosity measurements. The polymer coil radius and the volume per polymer coil were observed to decrease with the increase in the ratio of the lower alcohol in the solvent, whereas these values tended to decrease with the use of more hydrophilic lower alcohols. In addition, based on the results of the calculated entanglement index, it was found that when the hydrophobicity of the dispersed phase is reduced, the polymers were hardly entangled with each other. These results were significant, specifically when the ratio of the lower alcohol in the solvent was low. Estimation of the Pearson's correlation coefficients indicated that there were positive correlations between these indices and the mean volume diameter of PLLGA nanoparticles. This study shows that changing the composition of the dispersed phase, in which the PLLGA is dissolved, can change the conformation of the PLLGA molecules and control the size of the PLLGA nanoparticles.


Assuntos
Cloreto de Metileno/química , Nanopartículas/química , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Solventes/química , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Emulsões , Interações Hidrofóbicas e Hidrofílicas , Conformação Molecular , Psicoterapia Breve , Sonicação
10.
J Med Microbiol ; 69(8): 1062-1078, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32773005

RESUMO

Introduction. Acinetobacter baumannii is a critical priority pathogen listed by the World Health Organization due to increasing levels of resistance to carbapenem classes of antibiotics. It causes wound and other nosocomial infections, which can be life-threatening. Hence, there is an urgent need for the development of new classes of antibiotics.Aim. To study the interaction of carabapenems with class D beta-lactamases (oxacillinases) and analyse drug resistance by studying enzyme-substrate complexes using modelling approaches as a means of establishing correlations with the phenotypic data.Methodology. The three-dimensional structures of carbapenems (doripenem, ertapenem, imipenem and meropenem) were obtained from DrugBank and screened against class D beta-lactamases. Further, the study was extended with their variants. The variants' structure was homology-modelled using the Schrödinger Prime module (Schrödinger LLC, NY, USA).Results. The first discovered intrinsic beta-lactamase of Acinetobacter baumannii, OXA-51, had a binding energy value of -40.984 kcal mol-1, whereas other OXA-51 variants, such as OXA-64, OXA-110 and OXA-111, have values of -60.638, -66.756 and -67.751 kcal mol-1, respectively. The free energy values of OXA-51 variants produced better results than those of other groups.Conclusions. Imipenem and meropenem showed MIC values of 2 and 8 µg ml-1, respectively against OXA-51 in earlier studies, indicating that these are the most effective drugs for treatment of A. baumannii infection. According to our results, OXA-51 is an active enzyme that shows better interactions and is capable of hydrolyzing carbapenems. When correlating the hydrogen-bonding interaction with MIC values, the predicted results are in good agreement and might provide initial insights into performing similar studies related to OXA variants or other antibiotic-enzyme-based studies.


Assuntos
Acinetobacter baumannii/enzimologia , Carbapenêmicos/química , beta-Lactamases/química , Ampicilina/química , Carbapenêmicos/farmacologia , Domínio Catalítico , Simulação por Computador , Doripenem/química , Ertapenem/química , Hidrólise , Meropeném/química , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , beta-Lactamases/metabolismo
11.
Nucleic Acids Res ; 48(15): 8740-8754, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32644155

RESUMO

In mammalian cells, eight cytoplasmic aminoacyl-tRNA synthetases (AARS), and three non-synthetase proteins, reside in a large multi-tRNA synthetase complex (MSC). AARSs have critical roles in interpretation of the genetic code during protein synthesis, and in non-canonical functions unrelated to translation. Nonetheless, the structure and function of the MSC remain unclear. Partial or complete crystal structures of all MSC constituents have been reported; however, the structure of the holo-MSC has not been resolved. We have taken advantage of cross-linking mass spectrometry (XL-MS) and molecular docking to interrogate the three-dimensional architecture of the MSC in human HEK293T cells. The XL-MS approach uniquely provides structural information on flexibly appended domains, characteristic of nearly all MSC constituents. Using the MS-cleavable cross-linker, disuccinimidyl sulfoxide, inter-protein cross-links spanning all MSC constituents were observed, including cross-links between eight protein pairs not previously known to interact. Intra-protein cross-links defined new structural relationships between domains in several constituents. Unexpectedly, an asymmetric AARS distribution was observed featuring a clustering of tRNA anti-codon binding domains on one MSC face. Possibly, the non-uniform localization improves efficiency of delivery of charged tRNA's to an interacting ribosome during translation. In summary, we show a highly compact, 3D structural model of the human holo-MSC.


Assuntos
Aminoacil-tRNA Sintetases/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Conformação Proteica , Aminoacil-tRNA Sintetases/genética , Células HEK293 , Humanos , Espectrometria de Massas , Conformação Molecular , Simulação de Acoplamento Molecular , Complexos Multiproteicos/genética , Ligação Proteica
12.
Nucleic Acids Res ; 48(15): 8796-8807, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32652019

RESUMO

5-Formylcytosine (5fC) is a chemically edited, naturally occurring nucleobase which appears in the context of modified DNA strands. The understanding of the impact of 5fC on dsDNA physical properties is to date limited. In this work, we applied temperature-dependent 1H Chemical Exchange Saturation Transfer (CEST) NMR experiments to non-invasively and site-specifically measure the thermodynamic and kinetic influence of formylated cytosine nucleobase on the melting process involving dsDNA. Incorporation of 5fC within symmetrically positioned CpG sites destabilizes the whole dsDNA structure-as witnessed from the ∼2°C decrease in the melting temperature and 5-10 kJ mol-1 decrease in ΔG°-and affects the kinetic rates of association and dissociation. We observed an up to ∼5-fold enhancement of the dsDNA dissociation and an up to ∼3-fold reduction in ssDNA association rate constants, over multiple temperatures and for several proton reporters. Eyring and van't Hoff analysis proved that the destabilization is not localized, instead all base-pairs are affected and the transition states resembles the single-stranded conformation. These results advance our knowledge about the role of 5fC as a semi-permanent epigenetic modification and assist in the understanding of its interactions with reader proteins.


Assuntos
Citosina/análogos & derivados , DNA/efeitos dos fármacos , Conformação Molecular/efeitos dos fármacos , Termodinâmica , Pareamento de Bases/genética , Ilhas de CpG/genética , Citosina/química , Citosina/farmacologia , DNA/química , DNA/genética , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/genética , Cinética , Espectroscopia de Ressonância Magnética , Temperatura de Transição
13.
Nucleic Acids Res ; 48(15): 8675-8685, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32687167

RESUMO

Along with nucleobase pairing, base-base stacking interactions are one of the two main types of strong non-covalent interactions that define the unique secondary and tertiary structure of RNA. In this paper we studied two subfamilies of nucleobase-inserted stacking structures: (i) with any base intercalated between neighboring nucleotide residues (base-intercalated element, BIE, i + 1); (ii) with any base wedged into a hydrophobic cavity formed by heterocyclic bases of two nucleotides which are one nucleotide apart in sequence (base-wedged element, BWE, i + 2). We have exploited the growing database of natively folded RNA structures in Protein Data Bank to analyze the distribution and structural role of these motifs in RNA. We found that these structural elements initially found in yeast tRNAPhe are quite widespread among the tertiary structures of various RNAs. These motifs perform diverse roles in RNA 3D structure formation and its maintenance. They contribute to the folding of RNA bulges and loops and participate in long-range interactions of single-stranded stretches within RNA macromolecules. Furthermore, both base-intercalated and base-wedged motifs participate directly or indirectly in the formation of RNA functional centers, which interact with various ligands, antibiotics and proteins.


Assuntos
Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Proteínas de Ligação a RNA/ultraestrutura , RNA/ultraestrutura , Antibacterianos/química , Pareamento de Bases/genética , Substâncias Intercalantes/química , Ligantes , Modelos Moleculares , Conformação Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Nucleotídeos/química , Nucleotídeos/genética , RNA/química , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
14.
PLoS One ; 15(7): e0236420, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726324

RESUMO

Orientation analysis of fibers is widely applied in the fields of medical, material and life sciences. The orientation information allows predicting properties and behavior of materials to validate and guide a fabrication process of materials with controlled fiber orientation. Meanwhile, development of detector systems for high-resolution non-invasive 3D imaging techniques led to a significant increase in the amount of generated data per a sample up to dozens of gigabytes. Though plenty of 3D orientation estimation algorithms were developed in recent years, neither of them can process large datasets in a reasonable amount of time. This fact complicates the further analysis and makes impossible fast feedback to adjust fabrication parameters. In this work, we present a new method for quantifying the 3D orientation of fibers. The GPU implementation of the proposed method surpasses another popular method for 3D orientation analysis regarding accuracy and speed. The validation of both methods was performed on a synthetic dataset with varying parameters of fibers. Moreover, the proposed method was applied to perform orientation analysis of scaffolds with different fibrous micro-architecture studied with the synchrotron µCT imaging setup. Each acquired dataset of size 600x600x450 voxels was analyzed in less 2 minutes using standard PC equipped with a single GPU.


Assuntos
Sistemas Computacionais , Imageamento Tridimensional/métodos , Ciência dos Materiais/métodos , Conformação Molecular , Algoritmos
15.
Nat Commun ; 11(1): 3289, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620890

RESUMO

The spatial organization of chromosomes has key functional roles, yet how chromosomes fold remains poorly understood at the single-molecule level. Here, we employ models of polymer physics to investigate DNA loci in human HCT116 and IMR90 wild-type and cohesin depleted cells. Model predictions on single-molecule structures are validated against single-cell imaging data, providing evidence that chromosomal architecture is controlled by a thermodynamics mechanism of polymer phase separation whereby chromatin self-assembles in segregated globules by combinatorial interactions of chromatin factors that include CTCF and cohesin. The thermodynamics degeneracy of single-molecule conformations results in broad structural and temporal variability of TAD-like contact patterns. Globules establish stable environments where specific contacts are highly favored over stochastic encounters. Cohesin depletion reverses phase separation into randomly folded states, erasing average interaction patterns. Overall, globule phase separation appears to be a robust yet reversible mechanism of chromatin organization where stochasticity and specificity coexist.


Assuntos
Cromatina/química , Conformação Molecular , Fenômenos Físicos , Polímeros/química , Análise de Célula Única/métodos , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Células HCT116 , Humanos , Ligação Proteica , Processos Estocásticos , Termodinâmica
16.
BMC Bioinformatics ; 21(1): 272, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611376

RESUMO

BACKGROUND: Chromatin 3D conformation plays important roles in regulating gene or protein functions. High-throughout chromosome conformation capture (3C)-based technologies, such as Hi-C, have been exploited to acquire the contact frequencies among genomic loci at genome-scale. Various computational tools have been proposed to recover the underlying chromatin 3D structures from in situ Hi-C contact map data. As connected residuals in a polymer, neighboring genomic loci have intrinsic mutual dependencies in building a 3D conformation. However, current methods seldom take this feature into account. RESULTS: We present a method called ShNeigh, which combines the classical MDS technique with local dependence of neighboring loci modeled by a Gaussian formula, to infer the best 3D structure from noisy and incomplete contact frequency matrices. We validated ShNeigh by comparing it to two typical distance-based algorithms, ShRec3D and ChromSDE. The comparison results on simulated Hi-C dataset showed that, while keeping the high-speed nature of classical MDS, ShNeigh can recover the true structure better than ShRec3D and ChromSDE. Meanwhile, ShNeigh is more robust to data noise. On the publicly available human GM06990 Hi-C data, we demonstrated that the structures reconstructed by ShNeigh are more reproducible between different restriction enzymes than by ShRec3D and ChromSDE, especially at high resolutions manifested by sparse contact maps, which means ShNeigh is more robust to signal coverage. CONCLUSIONS: Our method can recover stable structures in high noise and sparse signal settings. It can also reconstruct similar structures from Hi-C data obtained using different restriction enzymes. Therefore, our method provides a new direction for enhancing the reconstruction quality of chromatin 3D structures.


Assuntos
Cromatina/química , Genômica/métodos , Algoritmos , Cromossomos/química , Cromossomos/genética , Loci Gênicos , Humanos , Conformação Molecular , Interface Usuário-Computador
17.
Comput Biol Med ; 122: 103849, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658736

RESUMO

SARS-CoV and SARS-CoV-2 do not appear to have functions of a hemagglutinin and neuraminidase. This is a mystery, because sugar binding activities appear essential to many other viruses including influenza and even most other coronaviruses in order to bind to and escape from the glycans (sugars, oligosaccharides or polysaccharides) characteristic of cell surfaces and saliva and mucin. The S1 N terminal Domains (S1-NTD) of the spike protein, largely responsible for the bulk of the characteristic knobs at the end of the spikes of SARS-CoV and SARS-CoV-2, are here predicted to be "hiding" sites for recognizing and binding glycans containing sialic acid. This may be important for infection and the ability of the virus to locate ACE2 as its known main host cell surface receptor, and if so it becomes a pharmaceutical target. It might even open up the possibility of an alternative receptor to ACE2. The prediction method developed, which uses amino acid residue sequence alone to predict domains or proteins that bind to sialic acids, is naïve, and will be advanced in future work. Nonetheless, it was surprising that such a very simple approach was so useful, and it can easily be reproduced in a very few lines of computer program to help make quick comparisons between SARS-CoV-2 sequences and to consider the effects of viral mutations.


Assuntos
Betacoronavirus/química , Biologia Computacional , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/química , Algoritmos , Motivos de Aminoácidos , Sítios de Ligação , Humanos , Conformação Molecular , Ácido N-Acetilneuramínico/química , Pandemias , Polissacarídeos/química , Vírus da SARS , Triptofano/química
18.
Nat Genet ; 52(7): 655-661, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32514124

RESUMO

Three-dimensional organization of the genome is important for transcriptional regulation1-7. In mammals, CTCF and the cohesin complex create submegabase structures with elevated internal chromatin contact frequencies, called topologically associating domains (TADs)8-12. Although TADs can contribute to transcriptional regulation, ablation of TAD organization by disrupting CTCF or the cohesin complex causes modest gene expression changes13-16. In contrast, CTCF is required for cell cycle regulation17, embryonic development and formation of various adult cell types18. To uncouple the role of CTCF in cell-state transitions and cell proliferation, we studied the effect of CTCF depletion during the conversion of human leukemic B cells into macrophages with minimal cell division. CTCF depletion disrupts TAD organization but not cell transdifferentiation. In contrast, CTCF depletion in induced macrophages impairs the full-blown upregulation of inflammatory genes after exposure to endotoxin. Our results demonstrate that CTCF-dependent genome topology is not strictly required for a functional cell-fate conversion but facilitates a rapid and efficient response to an external stimulus.


Assuntos
Linfócitos B/fisiologia , Fator de Ligação a CCCTC/fisiologia , Macrófagos/fisiologia , Mielopoese/fisiologia , Antígenos de Diferenciação/metabolismo , Fator de Ligação a CCCTC/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Cromatina/fisiologia , Regulação da Expressão Gênica , Humanos , Conformação Molecular , Mielopoese/genética , Conformação Proteica
19.
Food Chem ; 330: 127243, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32521403

RESUMO

The influences of konjac glucomannan (KGM) on the water distribution of frozen dough and steamed bread quaility made from frozen dough were addressed in this study. Low Field Nuclear Magnetic Resonance (LF-NMR) analysis demonstrated that the less tightly bound water of the dough was transferred to tightly bound water with the increase of KGM. Differential Scanning Calorimetry (DSC) showed that the freezable water of the frozen dough was transferred to non-freezable water with increasing the amount of KGM. DSC and LF-NMR results indicated the water state in the frozen dough was consistent. The addition of KGM enhanced the height and whiteness of steamed bread, but it decreased springiness, cohesiveness and resilience of steamed bread. As the KGM substitution rate increased, the hardness of steamed bread first decreased and then increased. The steamed bread with 1.5% of KGM showed the best quality.


Assuntos
Pão/análise , Mananas/química , Água/química , Varredura Diferencial de Calorimetria , Congelamento , Dureza , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Vapor
20.
Nat Commun ; 11(1): 3080, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32555154

RESUMO

The exact chemical structure of non-crystallising natural products is still one of the main challenges in Natural Sciences. Despite tremendous advances in total synthesis, the absolute structural determination of a myriad of natural products with very sensitive chemical functionalities remains undone. Here, we show that a metal-organic framework (MOF) with alcohol-containing arms and adsorbed water, enables selective hydrolysis of glycosyl bonds, supramolecular order with the so-formed chiral fragments and absolute determination of the organic structure by single-crystal X-ray crystallography in a single operation. This combined strategy based on a biomimetic, cheap, robust and multigram available solid catalyst opens the door to determine the absolute configuration of ketal compounds regardless degradation sensitiveness, and also to design extremely-mild metal-free solid-catalysed processes without formal acid protons.


Assuntos
Produtos Biológicos/química , Hidrolases/química , Estruturas Metalorgânicas/química , Adsorção , Álcoois/química , Catálise , Cristalografia por Raios X , Glicólise , Hidrólise , Cinética , Metais , Conformação Molecular , Estrutura Molecular , Porosidade , Relação Estrutura-Atividade , Difração de Raios X
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