RESUMO
The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum ß-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M ß-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum ß-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A ß-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.
Assuntos
Antibacterianos/farmacologia , Aztreonam/farmacologia , Monobactamas/farmacologia , beta-Lactamases/metabolismo , Antibacterianos/química , Aztreonam/química , Domínio Catalítico , Cristalografia por Raios X , Testes de Sensibilidade Microbiana , Monobactamas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , beta-Lactamases/genéticaRESUMO
In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.
Assuntos
Proteínas de Fluorescência Verde/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Amiloide/química , Bioensaio , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Temperatura Alta , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Eletricidade EstáticaRESUMO
Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.
Assuntos
Dosagem de Genes , Lentivirus/genética , Transdução Genética , Calibragem/normas , Técnicas de Transferência de Genes/normas , Vetores Genéticos/genética , Humanos , Células Jurkat , Mutagênese Insercional/genética , Padrões de Referência , Reprodutibilidade dos Testes , Transdução Genética/métodos , Transdução Genética/normas , Transfecção/métodos , Transfecção/normas , Estudos de Validação como Assunto , Integração Viral/genéticaRESUMO
The amino-acid sequence of the Toho-1 ß-lactamase contains several conserved residues in the active site, including Ser70, Lys73, Ser130 and Glu166, some of which coordinate a catalytic water molecule. This catalytic water molecule is essential in the acylation and deacylation parts of the reaction mechanism through which Toho-1 inactivates specific antibiotics and provides resistance to its expressing bacterial strains. To investigate the function of Glu166 in the acylation part of the catalytic mechanism, neutron and X-ray crystallographic studies were performed on a Glu166Gln mutant. The structure of this class A ß-lactamase mutant provides several insights into its previously reported reduced drug-binding kinetic rates. A joint refinement of both X-ray and neutron diffraction data was used to study the effects of the Glu166Gln mutation on the active site of Toho-1. This structure reveals that while the Glu166Gln mutation has a somewhat limited impact on the positions of the conserved amino acids within the active site, it displaces the catalytic water molecule from the active site. These subtle changes offer a structural explanation for the previously observed decreases in the binding of non-ß-lactam inhibitors such as the recently developed diazobicyclooctane inhibitor avibactam.
Assuntos
Proteínas de Bactérias/química , Cristalografia por Raios X/métodos , Ácido Glutâmico , Difração de Nêutrons/métodos , beta-Lactamases/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Antibacterianos/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Proteínas Mutantes/química , Água/químicaRESUMO
Protein dynamics are essential to function. One example of this is the various gating mechanisms within ion channels, which are transmembrane proteins that act as gateways into the cell. Typical ion channels switch between an open and closed state via a conformational transition which is often triggered by an external stimulus, such as ligand binding or pH and voltage differences. The atomic resolution structure of a potassium-selective ion channel named NaK2K has allowed us to observe that a hydro-phobic residue at the bottom of the selectivity filter, Phe92, appears in dual conformations. One of the two conformations of Phe92 restricts the diameter of the exit pore around the selectivity filter, limiting ion flow through the channel, while the other conformation of Phe92 provides a larger-diameter exit pore from the selectivity filter. Thus, it can be concluded that Phe92 acts as a hydro-phobic gate, regulating the flow of ions through the selectivity filter.
RESUMO
Martensitic transformations are the first-order crystal-to-crystal phase transitions that occur mostly in materials such as steel, alloys and ceramics, thus having many technological applications. These phase transitions are rarely observed in molecular crystals and have not been detected in protein crystals. Reversibly switchable fluorescent proteins are widely used in biotechnology, including super-resolution molecular imaging, and hold promise as candidate biomaterials for future high-tech applications. Here, we report on a reversibly switchable fluorescent protein, Tetdron, whose crystals undergo a photo-induced martensitic transformation at room temperature. Room-temperature X-ray crystallography demonstrates that at equilibrium Tetdron chromophores are all in the trans configuration, with an â¼1:1 mixture of their protonated and deprotonated forms. Irradiation of a Tetdron crystal with 400â nm light induces a martensitic transformation, which results in Tetdron tetramerization at room temperature revealed by X-ray photocrystallography. Crystal and solution spectroscopic measurements provide evidence that the photo-induced martensitic phase transition is coupled with the chromophore deprotonation, but no trans-cis isomerization is detected in the structure of an irradiated crystal. It is hypothesized that protein dynamics assists in the light-induced proton transfer from the chromophore to the bulk solvent and in the ensuing martensitic phase transition. The unique properties of Tetdron may be useful in developing novel biomaterials for optogenetics, data storage and nanotechnology.
RESUMO
[This retracts the article DOI: 10.1107/S2052252519005761.].
RESUMO
The mechanism by which potassium ions are transported through ion channels is currently being investigated by several groups using many different techniques. Clarification of the location of water molecules during transport is central to understanding how these integral membrane proteins function. Neutrons have a unique sensitivity to both hydrogen and potassium, rendering neutron crystallography capable of distinguishing waters from K+ ions. Here, the collection of a complete neutron data set from a potassium ion channel to a resolution of 3.55â Å using the Macromolecular Neutron Diffractometer (MaNDi) is reported. A room-temperature X-ray data set was also collected from the same crystal to a resolution of 2.50â Å. Upon further refinement, these results will help to further clarify the ion/water population within the selectivity filter of potassium ion channels.
Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/química , Nêutrons , Canais de Potássio/química , Cristalografia por Raios X , Modelos Moleculares , Conformação ProteicaRESUMO
Crystallography is the powerhouse technique for molecular structure determination, with applications in fields ranging from energy storage to drug design. Accurate structure determination, however, relies partly on determining the precise locations and integrated intensities of Bragg peaks in the resulting data. Here, we describe a method for Bragg peak integration that is accomplished using neural networks. The network is based on a U-Net and identifies peaks in three-dimensional reciprocal space through segmentation, allowing prediction of the full 3D peak shape from noisy data that is commonly difficult to process. The procedure for generating appropriate training sets is detailed. Trained networks achieve Dice coefficients of 0.82 and mean IoUs of 0.69. Carrying out integration over entire datasets, it is demonstrated that integrating neural network-predicted peaks results in improved intensity statistics. Furthermore, using a second dataset, the possibility of transfer learning between datasets is shown. Given the ubiquity and growing complexity of crystallography, we anticipate integration by machine learning to play an increasingly important role across the physical sciences. These early results demonstrate the applicability of deep learning techniques for integrating crystallography data and suggest a possible role in the next generation of crystallography experiments.
RESUMO
Neutron crystallography offers enormous potential to complement structures from X-ray crystallography by clarifying the positions of low-Z elements, namely hydrogen. Macromolecular neutron crystallography, however, remains limited, in part owing to the challenge of integrating peak shapes from pulsed-source experiments. To advance existing software, this article demonstrates the use of machine learning to refine peak locations, predict peak shapes and yield more accurate integrated intensities when applied to whole data sets from a protein crystal. The artificial neural network, based on the U-Net architecture commonly used for image segmentation, is trained using about 100â 000 simulated training peaks derived from strong peaks. After 100 training epochs (a round of training over the whole data set broken into smaller batches), training converges and achieves a Dice coefficient of around 65%, in contrast to just 15% for negative control data sets. Integrating whole peak sets using the neural network yields improved intensity statistics compared with other integration methods, including k-nearest neighbours. These results demonstrate, for the first time, that neural networks can learn peak shapes and be used to integrate Bragg peaks. It is expected that integration using neural networks can be further developed to increase the quality of neutron, electron and X-ray crystallography data.
RESUMO
The use of boiled-off liquid nitrogen to maintain protein crystals at 100â K during X-ray data collection has become almost universal. Applying this to neutron protein crystallography offers the opportunity to significantly broaden the scope of biochemical problems that can be addressed, although care must be taken in assuming that direct extrapolation to room temperature is always valid. Here, the history to date of neutron protein cryo-crystallography and the particular problems and solutions associated with the mounting and cryocooling of the larger crystals needed for neutron crystallography are reviewed. Finally, the outlook for further cryogenic neutron studies using existing and future neutron instrumentation is discussed.
Assuntos
Temperatura Baixa , Difração de Nêutrons/métodos , Proteínas/química , Cristalografia , História do Século XX , História do Século XXI , Difração de Nêutrons/históriaRESUMO
Neutron crystallography is a powerful technique for directly visualizing the locations of H atoms in biological macromolecules. This information has provided key new insights into enzyme mechanisms, ligand binding and hydration. However, despite the importance of this information, the application of neutron crystallography in biology has been limited by the relatively low flux of available neutron beams and the large incoherent neutron scattering from hydrogen, both of which contribute to weak diffraction data with relatively low signal-to-background ratios. A method has been developed to fit weak data based on three-dimensional profile fitting of Bragg peaks in reciprocal space by an Ikeda-Carpenter function with a bivariate Gaussian. When applied to data collected from three different proteins, three-dimensional profile fitting yields intensities with higher correlation coefficients (CC1/2) at high resolutions, decreased Rfree factors, extended resolutions and improved nuclear density maps. Importantly, additional features are revealed in nuclear density maps that may provide additional scientific information. These results suggest that three-dimensional profile fitting will help to extend the capabilities of neutron macromolecular crystallography.
Assuntos
Difração de Nêutrons/métodos , Conformação Proteica , Proteínas/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Nêutrons , Proteínas/metabolismo , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Potassium ion channels utilize a highly selective filter to rapidly transport K+ ions across cellular membranes. This selectivity filter is composed of four binding sites which display almost equal electron density in crystal structures with high potassium ion concentrations. This electron density can be interpreted to reflect a superposition of alternating potassium ion and water occupied states or as adjacent potassium ions. Here, we use single wavelength anomalous dispersion (SAD) X-ray diffraction data collected near the potassium absorption edge to show experimentally that all ion binding sites within the selectivity filter are fully occupied by K+ ions. These data support the hypothesis that potassium ion transport occurs by direct Coulomb knock-on, and provide an example of solving the phase problem by K-SAD.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Difração de Raios X , Bacillus cereus/metabolismo , Transporte de ÍonsRESUMO
The role of the conserved residue Tyr105 in class A ß-lactamases has been the subject of investigation using both structural studies and saturation mutagenesis. Both have shown that while it does not need to be strictly conserved for activity, it is important for substrate recognition. With this in mind we determined the crystal structure of Toho1 ß-lactamase at 15 K to 1.10 Å resolution in complex with penicillin. As expected a ring-opened penicillin molecule bound to Ser70 the catalytic nucleophile, can clearly be seen in electron density in the active site. In addition to the trapped penicillin, however, are two additional intact ring-closed penicillin molecules, captured by the enzyme through noncovalent interactions at the edge of the active site.
RESUMO
We report the engineering of a new reversibly switching chromogenic protein, Dathail. Dathail was evolved from the extremely thermostable fluorescent proteins thermal green protein (TGP) and eCGP123 using directed evolution and ratiometric sorting. Dathail has two spectrally distinct chromogenic states with low quantum yields, corresponding to absorbance in a ground state with a maximum at 389nm, and a photo-induced metastable state with a maximum at 497nm. In contrast to all previously described photoswitchable proteins, both spectral states of Dathail are non-fluorescent. The photo-induced chromogenic state of Dathail has a lifetime of ~50min at 293K and pH7.5 as measured by UV-Vis spectrophotometry, returning to the ground state through thermal relaxation. X-ray crystallography provided structural insights supporting a change in conformation and coordination in the chromophore pocket as being responsible for Dathail's photoswitching. Neutron crystallography, carried out for the first time on a protein from the green fluorescent protein family, showed a distribution of hydrogen atoms revealing protonation of the chromophore 4-hydroxybenzyl group in the ground state. The neutron structure also supports the hypothesis that the photo-induced proton transfer from the chromophore occurs through water-mediated proton relay into the bulk solvent. Beyond its spectroscopic curiosity, Dathail has several characteristics that are improvements for applications, including low background fluorescence, large spectral separation, rapid switching time, and the ability to switch many times. Therefore, Dathail is likely to be extremely useful in the quickly developing fields of imaging and biosensors, including photochromic Förster resonance energy transfer, high-resolution microscopy, and live tracking within the cell.