RESUMO
Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B12) can act as a light-sensing chromophore heralded a new field of B12-photobiology. Although microbial genome analysis indicates that photoactive B12-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.
Assuntos
Pigmentos Biliares , Fotorreceptores Microbianos , Fotoquímica , Biliverdina , Proteínas de Bactérias/metabolismo , Fotorreceptores Microbianos/química , LuzRESUMO
In this review, we describe recent research developments into radiation damage effects in macromolecular X-ray crystallography observed at synchrotrons and X-ray free electron lasers. Radiation damage in small molecule X-ray crystallography, small angle X-ray scattering experiments, microelectron diffraction, and single particle cryo-electron microscopy is briefly covered.
Assuntos
Elétrons , Síncrotrons , Microscopia Crioeletrônica , Cristalografia por Raios X , Raios X , Difração de Raios XRESUMO
An understanding of radiation damage effects suffered by biological samples during structural analysis using both X-rays and electrons is pivotal to obtain reliable molecular models of imaged molecules. This special issue on radiation damage contains six papers reporting analyses of damage from a range of biophysical imaging techniques. For X-ray diffraction, an in-depth study of multi-crystal small-wedge data collection single-wavelength anomalous dispersion phasing protocols is presented, concluding that an absorbed dose of 5â MGy per crystal was optimal to allow reliable phasing. For small-angle X-ray scattering, experiments are reported that evaluate the efficacy of three radical scavengers using a protein designed to give a clear signature of damage in the form of a large conformational change upon the breakage of a disulfide bond. The use of X-rays to induce OH radicals from the radiolysis of water for X-ray footprinting are covered in two papers. In the first, new developments and the data collection pipeline at the NSLS-II high-throughput dedicated synchrotron beamline are described, and, in the second, the X-ray induced changes in three different proteins under aerobic and low-oxygen conditions are investigated and correlated with the absorbed dose. Studies in XFEL science are represented by a report on simulations of ultrafast dynamics in protic ionic liquids, and, lastly, a broad coverage of possible methods for dose efficiency improvement in modalities using electrons is presented. These papers, as well as a brief synopsis of some other relevant literature published since the last Journal of Synchrotron Radiation Special Issue on Radiation Damage in 2019, are summarized below.
Assuntos
Substâncias Macromoleculares/química , Substâncias Macromoleculares/efeitos da radiação , Biofísica , Cristalografia por Raios X , Elétrons , Doses de Radiação , Lesões por Radiação , Espalhamento de Radiação , Síncrotrons , Difração de Raios XRESUMO
Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment, and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here, we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, in which the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced x-ray emission and x-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.
Assuntos
Amiloide , Zinco , Humanos , Insulina , Cinética , Espectroscopia por Absorção de Raios XRESUMO
Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses (D1/2) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.
Assuntos
Cristalografia por Raios X/métodos , Muramidase/química , Síncrotrons , Temperatura , CristalizaçãoRESUMO
With the continuing development of beamlines for macromolecular crystallography (MX) over the last few years providing ever higher X-ray flux densities, it has become even more important to be aware of the effects of radiation damage on the resulting structures. Nine papers in this issue cover a range of aspects related to the physics and chemistry of the manifestations of this damage, as observed in both MX and small-angle X-ray scattering (SAXS) on crystals, solutions and tissue samples. The reports include measurements of the heating caused by X-ray irradiation in ruby microcrystals, low-dose experiments examining damage rates as a function of incident X-ray energy up to 30â keV on a metallo-enzyme using a CdTe detector of high quantum efficiency as well as a theoretical analysis of the gains predicted in diffraction efficiency using these detectors, a SAXS examination of low-dose radiation exposure effects on the dissociation of a protein complex related to human health, theoretical calculations describing radiation chemistry pathways which aim to explain the specific structural damage widely observed in proteins, investigation of radiation-induced damage effects in a DNA crystal, a case study on a metallo-enzyme where structural movements thought to be mechanism related might actually be radiation-damage-induced changes, and finally a review describing what X-ray radiation-induced cysteine modifications can teach us about protein dynamics and catalysis. These papers, along with some other relevant literature published since the last Journal of Synchrotron Radiation Radiation Damage special issue in 2017, are briefly summarized below.
Assuntos
Dano ao DNA , Síncrotrons , Raios X , Compostos de Cádmio/química , Cristalização , DNA/química , DNA/efeitos da radiação , Espalhamento a Baixo Ângulo , Telúrio/química , Difração de Raios XRESUMO
Radiation damage inflicted on macromolecular crystals during X-ray diffraction experiments remains a limiting factor for structure solution, even when samples are cooled to cryotemperatures (~100 K). Efforts to establish mitigation strategies are ongoing and various approaches, summarized below, have been investigated over the last 15 years, resulting in a deeper understanding of the physical and chemical factors affecting damage rates. The recent advent of X-ray free electron lasers permits "diffraction-before-destruction" by providing highly brilliant and short (a few tens of fs) X-ray pulses. New fourth generation synchrotron sources now coming on line with higher X-ray flux densities than those available from third generation synchrotrons will bring the issue of radiation damage once more to the fore for structural biologists.
Assuntos
Cristalografia por Raios X/métodos , Elétrons , Substâncias Macromoleculares/ultraestrutura , Proteínas/ultraestrutura , Síncrotrons/instrumentação , Cristalização , Sequestradores de Radicais Livres/química , Radicais Livres/antagonistas & inibidores , Lasers , Substâncias Macromoleculares/química , Conformação Proteica , Proteínas/química , Doses de Radiação , Raios XRESUMO
Despite significant progress made over more than 15 years of research, structural biologists are still grappling with the issue of radiation damage suffered by macromolecular crystals which is induced by the resultant radiation chemistry occurring during X-ray diffraction experiments. Further insights into these effects and the possible mitigation strategies for use in both diffraction and SAXS experiments are given in eight papers in this volume. In particular, damage during experimental phasing is addressed, scavengers for SAXS experiments are investigated, microcrystals are imaged, data collection strategies are optimized, specific damage to tyrosine residues is reexamined, and room temperature conformational heterogeneity as a function of dose is explored. The brief summary below puts these papers into perspective relative to other ongoing radiation damage research on macromolecules.
Assuntos
Cristalografia por Raios X , Substâncias Macromoleculares , Espalhamento a Baixo Ângulo , Difração de Raios X , Raios XRESUMO
Radiation damage induced by X-ray beams during macromolecular diffraction experiments remains an issue of concern in structural biology. While advances in our understanding of this phenomenon, driven in part by a series of workshops in this area, undoubtedly have been and are still being made, there are still questions to be answered. Eight papers in this volume give a flavour of ongoing investigations, addressing various issues. These range over: a proposed new metric derived from atomic B-factors for identifying potentially damaged amino acid residues, a study of the relative damage susceptibility of protein and DNA in a DNA/protein complex, a report of an indication of specific radiation damage to a protein determined from data collected using an X-ray free-electron laser (FEL), an account of the challenges in FEL raw diffraction data analysis, an exploration of the possibilities of using radiation damage induced phasing to solve structures using FELs, simulations of radiation damage as a function of FEL temporal pulse profiles, results on the influence of radiation damage during scanning X-ray diffraction measurements and, lastly, consideration of strategies for minimizing radiation damage during SAXS experiments. In this short introduction, these contributions are briefly placed in the context of other current work on radiation damage in the field.
Assuntos
Substâncias Macromoleculares/efeitos da radiação , Lesões por Radiação , Difração de Raios X/métodos , Simulação por Computador , Cristalografia por Raios X , Humanos , Modelos Moleculares , Espalhamento a Baixo Ângulo , Sensibilidade e EspecificidadeRESUMO
X-ray-radiation-induced alterations to protein structures are still a severe problem in macromolecular crystallography. One way to avoid the influence of radiation damage is to reduce the X-ray dose absorbed by the crystal during data collection. However, here it is demonstrated using the example of the membrane protein bacteriorhodopsin (bR) that even a low dose of less than 0.06â MGy may induce structural alterations in proteins. This dose is about 500 times smaller than the experimental dose limit which should ideally not be exceeded per data set (i.e. 30â MGy) and 20 times smaller than previously detected specific radiation damage at the bR active site. To date, it is the lowest dose at which radiation modification of a protein structure has been described. Complementary use was made of high-resolution X-ray crystallography and online microspectrophotometry to quantitatively study low-dose X-ray-induced changes. It is shown that structural changes of the protein correlate with the spectroscopically observed formation of the so-called bR orange species. Evidence is provided for structural modifications taking place at the protein active site that should be taken into account in crystallographic studies which aim to elucidate the molecular mechanisms of bR function.
Assuntos
Bacteriorodopsinas/química , Cristalografia por Raios X/métodos , Proteínas/química , Proteínas/efeitos da radiação , Raios X , Domínio Catalítico , Relação Dose-Resposta à Radiação , Análise de Fourier , Modelos Moleculares , Conformação ProteicaRESUMO
Research into radiation damage in macromolecular crystallography has matured over the last few years, resulting in a better understanding of both the processes and timescales involved. In turn this is now allowing practical recommendations for the optimization of crystal dose lifetime to be suggested. Some long-standing questions have been answered by recent investigations, and from these answers new challenges arise and areas of investigation can be proposed. Six papers published in this volume give an indication of some of the current directions of this field and also that of single-particle cryo-microscopy, and the brief summary below places them into the overall framework of ongoing research into macromolecular crystallography radiation damage.
Assuntos
Substâncias Macromoleculares/efeitos da radiação , Cristalografia por Raios X , Substâncias Macromoleculares/química , Microscopia Eletrônica , Raios Ultravioleta , Raios XRESUMO
Radiation damage in macromolecular crystallography has become a mainstream concern over the last ten years. The current status of research into this area is briefly assessed, and the ten new papers published in this issue are set into the context of previous work in the field. Some novel and exciting developments emerging over the last two years are also summarized.
Assuntos
Lesões por Radiação , Cristalografia por Raios XRESUMO
The temperature-dependence of radiation damage in macromolecular X-ray crystallography is currently much debated. Most protein crystallographic studies are based on data collected at 100 K. Data collection at temperatures below 100 K has been proposed to reduce radiation damage and above 100 K to be useful for kinetic crystallography that is aimed at the generation and trapping of protein intermediate states. Here the global and specific synchrotron-radiation sensitivity of crystalline thermolysin at 100 and 160 K are compared. Both types of damage are higher at 160 K than at 100 K. At 160 K more residue types are affected (Lys, Asp, Gln, Pro, Thr, Met, Asn) than at 100 K (Met, Asp, Glu, Lys). The X-ray-induced relative atomic B-factor increase is shown to correlate with the proximity of the atom to the nearest solvent channel at 160 K. Two models may explain the observed correlation: either an increase in static disorder or an increased attack of hydroxyl radicals from the solvent area of the crystal.
RESUMO
The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.
Assuntos
Inibidores da Colinesterase/química , Colinesterases/química , Cloreto de Mercúrio/química , Acetilcolinesterase/química , Acetilcolinesterase/genética , Animais , Sítios de Ligação , Butirilcolinesterase/química , Butirilcolinesterase/genética , Colinesterases/genética , Cristalografia por Raios X , Cisteína/química , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Electrophorus/genética , Electrophorus/metabolismo , Humanos , Cinética , Luz , Modelos Químicos , Modelos Moleculares , Nitrobenzenos/química , Fenilacetatos/química , Conformação Proteica , Proteínas Recombinantes/química , Espalhamento de Radiação , Torpedo/genética , Torpedo/metabolismoRESUMO
Intense synchrotron radiation produces specific structural and chemical damage to crystalline proteins even at 100 K. Carboxyl groups of acidic residues (Glu, Asp) losing their definition is one of the major effects observed. Here, the susceptibilities to X-ray damage of acidic residues in tetrameric malate dehydrogenase from Haloarcula marismortui are investigated. The marked excess of acidic residues in this halophilic enzyme makes it an ideal target to determine how specific damage to acidic residues is related to their structural and chemical environment. Four conclusions are drawn. (i) Acidic residues interacting with the side-chains of lysine and arginine residues are less affected by radiation damage than those interacting with serine, threonine and tyrosine side-chains. This suggests that residues with higher pK(a) values are more vulnerable to damage than those with a lower pK(a). However, such a correlation was not found when calculated pK(a) values were inspected. (ii) Acidic side-chains located in the enzymatic active site are the most radiation-sensitive ones. (iii) Acidic residues in the internal cavity formed by the four monomers and those involved in crystal contacts appear to be particularly susceptible. (iv) No correlation was found between radiation susceptibility and solvent accessibility.
Assuntos
Aminoácidos/química , Aminoácidos/efeitos da radiação , Cristalografia por Raios X/métodos , Haloarcula marismortui/química , Malato Desidrogenase/química , Malato Desidrogenase/efeitos da radiação , Modelos Químicos , Simulação por Computador , Relação Dose-Resposta à Radiação , Concentração de Íons de Hidrogênio , Malato Desidrogenase/ultraestrutura , Modelos Moleculares , Conformação Proteica/efeitos da radiação , Desnaturação Proteica/efeitos da radiação , Doses de Radiação , Soluções , Solventes/química , Relação Estrutura-Atividade , Raios XRESUMO
Caged compounds in combination with protein crystallography represent a valuable tool in studies of enzyme reaction intermediates. To date, photochemical triggering of reactions has been performed close to room temperature. Synchronous reaction initiation has only been achieved with enzymes of relatively slow turnover (<0.1 s(-1)) and caged compounds of high quantum yield. Here X-ray crystallography and microspectrophotometry were used to provide evidence that (nitrophenyl)ethyl (NPE) ester bonds can be photolyzed by UV light at cryotemperatures. NPE-caged ATP in flash-cooled crystals of Mycobacterium tuberculosis thymidylate kinase was photolyzed successfully at 100-150 K as assessed by the structural observation of ATP-dependent enzymatic conversion of TMP to TDP after temporarily warming the crystals to room temperature. A new method is proposed in which cryo-photolysis combined with temperature-controlled protein crystallography can be used to trap reaction intermediates even in some of the fastest enzymes and/or when only compounds of low quantum yield are available. Raising the temperature after cryophotolysis may allow a transition barrier to be passed and an intermediate to accumulate in the crystal. A comparable method has only been used so far with proteins displaying endogenous photosensitivity. The approach described here opens the way to studying the reaction mechanisms of a much larger number of crystalline enzymes. Furthermore, it is shown that X-ray-induced radiolysis of caged compounds occurs if high-intensity synchrotron beamlines are used. This caveat should be taken into account when deriving data-collection protocols. It could also be used potentially as a way to trigger reactions.