RESUMEN
Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.
RESUMEN
Catalase is an antioxidant enzyme that breaks down hydrogen peroxide (H2O2) into molecular oxygen and water. In all monofunctional catalases the pathway that H2O2 takes to the catalytic centre is via the `main channel'. However, the structure of this channel differs in large-subunit and small-subunit catalases. In large-subunit catalases the channel is 15â Å longer and consists of two distinct parts, including a hydrophobic lower region near the heme and a hydrophilic upper region where multiple H2O2 routes are possible. Conserved glutamic acid and threonine residues are located near the intersection of these two regions. Mutations of these two residues in the Scytalidium thermophilum catalase had no significant effect on catalase activity. However, the secondary phenol oxidase activity was markedly altered, with kcat and kcat/Km values that were significantly increased in the five variants E484A, E484I, T188D, T188I and T188F. These variants also showed a lower affinity for inhibitors of oxidase activity than the wild-type enzyme and a higher affinity for phenolic substrates. Oxidation of heme b to heme d did not occur in most of the studied variants. Structural changes in solvent-chain integrity and channel architecture were also observed. In summary, modification of the main-channel gate glutamic acid and threonine residues has a greater influence on the secondary activity of the catalase enzyme, and the oxidation of heme b to heme d is predominantly inhibited by their conversion to aliphatic and aromatic residues.
Asunto(s)
Ácido Glutámico , Peróxido de Hidrógeno , Catalasa/química , Peróxido de Hidrógeno/química , Hemo/química , TreoninaRESUMEN
Incoherent neutron spectroscopy, in combination with dynamic light scattering, was used to investigate the effect of ligand binding on the center-of-mass self-diffusion and internal diffusive dynamics of Escherichia coli aspartate α-decarboxylase (ADC). The X-ray crystal structure of ADC in complex with the D-serine inhibitor was also determined, and molecular dynamics simulations were used to further probe the structural rearrangements that occur as a result of ligand binding. These experiments reveal that D-serine forms hydrogen bonds with some of the active site residues, that higher order oligomers of the ADC tetramer exist on ns-ms time-scales, and also show that ligand binding both affects the ADC internal diffusive dynamics and appears to further increase the size of the higher order oligomers.
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Ácido Aspártico , Carboxiliasas/metabolismo , Serina , Difusión , Escherichia coli , Ligandos , Modelos MolecularesRESUMEN
With recent developments in X-ray sources, instrumentation and data-analysis tools, time-resolved crystallographic experiments, which were originally the preserve of a few expert groups, are becoming simpler and can be carried out at more radiation sources, and are thus increasingly accessible to a growing user base. However, these experiments are just that: discrete experiments, not just `data collections'. As such, careful planning and consideration of potential pitfalls is required to enable a successful experiment. Here, some of the key factors that should be considered during the planning and execution of a time-resolved structural study are outlined, with a particular focus on synchrotron-based experiments.
Asunto(s)
Cristalografía por Rayos X/instrumentación , Cristalografía por Rayos X/métodos , Sincrotrones , Animales , Análisis de Datos , Enzimas/química , HumanosRESUMEN
Scytalidium catalase is a homotetramer including heme d in each subunit. Its primary function is the dismutation of H2O2 to water and oxygen, but it is also able to oxidase various small organic compounds including catechol and phenol. The crystal structure of Scytalidium catalase reveals the presence of three linked channels providing access to the exterior like other catalases reported so far. The function of these channels has been extensively studied, revealing the possible routes for substrate flow and product release. In this report, we have focussed on the semi-conserved residue Val228, located near to the vinyl groups of the heme at the opening of the lateral channel. Its replacement with Ala, Ser, Gly, Cys, Phe and Ile were tested. We observed a significant decrease in catalytic efficiency in all mutants with the exception of a remarkable increase in oxidase activity when Val228 was mutated to either Ala, Gly or Ser. The reduced catalytic efficiencies are characterized in terms of the restriction of hydrogen peroxide as electron acceptor in the active centre resulting from the opening of lateral channel inlet by introducing the smaller side chain residues. On the other hand, the increased oxidase activity is explained by allowing the suitable electron donor to approach more closely to the heme. The crystal structures of V228C and V228I were determined at 1.41 and 1.47 Å resolution, respectively. The lateral channels of the V228C and V228I presented a broadly identical chain of arranged waters to that observed for wild-type enzyme.
Asunto(s)
Catalasa/genética , Hemo/química , Sordariales/enzimología , Sordariales/genética , Ascomicetos/enzimología , Ascomicetos/genética , Catalasa/química , Catalasa/metabolismo , Catálisis , Dominio Catalítico , Hemo/análogos & derivados , Peróxido de Hidrógeno/química , Modelos Moleculares , Sordariales/metabolismoRESUMEN
Photoactivatable fluorescent proteins (PA-FPs) are a powerful non-invasive tool in high-resolution live-cell imaging. They can be converted from an inactive to an active form by light, enabling the spatial and temporal trafficking of proteins and cell dynamics. PA-FPs have been previously generated by mutating selected residues in the chromophore or in its close proximity. A new strategy to generate PA-FPs is the genetic incorporation of unnatural amino acids (UAAs) containing photocaged groups using unique suppressor tRNA/aminoacyl-tRNA synthetase pairs. We set out to develop a photoactivatable GFP variant suitable for time-resolved structural studies. Here, we report the crystal structure of superfolder GFP (sfGFP) containing the UAA ortho-nitrobenzyl-tyrosine (ONBY) at position 66 and its spectroscopic characterization. Surprisingly, the crystal structure (to 2.7 Å resolution) reveals a dimeric domain-swapped arrangement of sfGFP66ONBY with residues 1-142 of one molecule associating with residues 148-234 from another molecule. This unusual domain-swapped structure supports a previously postulated GFP folding pathway that proceeds via an equilibrium intermediate.
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Aminoácidos/genética , Proteínas Fluorescentes Verdes/química , Conformación Proteica , Pliegue de Proteína , Aminoácidos/química , Aminoacil-ARNt Sintetasas/genética , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas Fluorescentes Verdes/genética , Methanocaldococcus/genética , Imagen Molecular/métodos , Mutación/genética , Fenilalanina/química , ARN de Transferencia/química , ARN de Transferencia/genética , Tirosina/genéticaRESUMEN
We report a new synthetic route to a series of α-carboxynitrobenzyl photocaged l-aspartates for application in time-resolved structural biology. The resulting compounds were characterised in terms of UV/Vis absorption properties, aqueous solubility and stability, and photocleavage rates (τ = µs to ms) and quantum yields (φ = 0.05 to 0.14).
RESUMEN
The catalase from Scytalidium thermophilum is a homotetramer containing a heme d in each active site. Although the enzyme has a classical monofunctional catalase fold, it also possesses oxidase activity towards a number of small organics, including catechol and phenol. In order to further investigate this, the crystal structure of the complex of the catalase with the classical catalase inhibitor 3-amino-1,2,4-triazole (3TR) was determined at 1.95â Å resolution. Surprisingly, no binding to the heme site was observed; instead, 3TR occupies a binding site corresponding to the NADPH-binding pocket in mammalian catalases at the entrance to a lateral channel leading to the heme. Kinetic analysis of site-directed mutants supports the assignment of this pocket as the binding site for oxidase substrates.
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Sitios de Unión , Catalasa/química , Proteínas Fúngicas/química , Hongos/enzimología , Amitrol (Herbicida)/metabolismo , Catalasa/antagonistas & inhibidores , Dominio Catalítico , Cristalografía por Rayos X , Hemo/análogos & derivados , Hemo/metabolismo , NADP/metabolismo , Oxidorreductasas/metabolismoRESUMEN
A new method of performing pump-probe experiments is proposed and experimentally demonstrated by a proof of concept on the millisecond scale. The idea behind this method is to measure the total probe intensity arising from several time points as a group, instead of measuring each time separately. These measurements are multiplexes that are then transformed into the true signal via multiplication with a binary Hadamard S matrix. Each group of probe pulses is determined by using the pattern of a row of the Hadamard S matrix and the experiment is completed by rotating this pattern by one step for each sample excitation until the original pattern is again produced. Thus to measure n time points, n excitation events are needed and n probe patterns each taken from the n × n S matrix. The time resolution is determined by the shortest time between the probe pulses. In principle, this method could be used over all timescales, instead of the conventional pump-probe method which uses delay lines for picosecond and faster time resolution, or fast detectors and oscilloscopes on longer timescales. This new method is particularly suitable for situations where the probe intensity is weak and/or the detector is noisy. When the detector is noisy, there is in principle a signal to noise advantage over conventional pump-probe methods.
RESUMEN
Time-resolved structural information is key to understand the mechanism of biological processes, such as catalysis and signalling. Recent developments in X-ray sources as well as data collection and analysis methods are making routine time-resolved X-ray crystallography and solution scattering experiments a real possibility for structural biologists. Here we review the information that can be obtained from these techniques and discuss the considerations that must be taken into account when designing a time-resolved experiment.
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Cristalografía por Rayos X/métodos , Electrones , Rayos Láser , Dispersión de Radiación , Sincrotrones , Cristalografía por Rayos X/instrumentación , Humanos , SolucionesRESUMEN
We describe a method for performing time-resolved X-ray crystallographic experiments based on the Hadamard transform, in which time resolution is defined by the underlying periodicity of the probe pulse sequence, and signal/noise is greatly improved over that for the fastest pump-probe experiments depending on a single pulse. This approach should be applicable on standard synchrotron beamlines and will enable high-resolution measurements of protein and small-molecule structural dynamics. It is also applicable to other time-resolved measurements where a probe can be encoded, such as pump-probe spectroscopy.
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Cristalografía por Rayos X/métodos , Proteínas de Plantas/química , Conformación Proteica , Relación Señal-Ruido , Factores de TiempoRESUMEN
Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of ß-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.
Asunto(s)
Escherichia coli/enzimología , Glutamato Descarboxilasa/química , Activación Enzimática , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Modelos Moleculares , Mutación , Estructura Terciaria de Proteína , Treonina/genética , Treonina/metabolismoRESUMEN
During X-ray irradiation protein crystals radiate energy in the form of small amounts of visible light. This is known as X-ray-excited optical luminescence (XEOL). The XEOL of several proteins and their constituent amino acids has been characterized using the microspectrophotometers at the Swiss Light Source and Diamond Light Source. XEOL arises primarily from aromatic amino acids, but the effects of local environment and quenching within a crystal mean that the XEOL spectrum of a crystal is not the simple sum of the spectra of its constituent parts. Upon repeated exposure to X-rays XEOL spectra decay non-uniformly, suggesting that XEOL is sensitive to site-specific radiation damage. However, rates of XEOL decay were found not to correlate to decays in diffracting power, making XEOL of limited use as a metric for radiation damage to protein crystals.
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Aminoácidos/química , Proteínas/química , Difracción de Rayos X/métodos , Animales , Apoferritinas/química , Pollos , Cristalización , Escherichia coli/enzimología , Caballos , Luminiscencia , Muramidasa/química , Oxo-Ácido-Liasas/química , Termolisina/química , Rayos XRESUMEN
The structural information and functional insight obtained from X-ray crystallography can be enhanced by the use of complementary spectroscopies. Here the information that can be obtained from spectroscopic methods commonly used in conjunction with X-ray crystallography and best-practice single-crystal UV-Vis absorption data collection are briefly reviewed. Using data collected with the in situ system at the Swiss Light Source, the time and dose scales of low-dose X-ray-induced radiation damage and solvated electron generation in metalloproteins at 100 K are investigated. The effect of dose rate on these scales is also discussed.