RESUMEN
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.
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Cristalografía por Rayos X/métodos , Muramidasa/química , Sincrotrones , Temperatura , CristalizaciónRESUMEN
Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined inâ vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both inâ vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.
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Escherichia coli , Microscopía , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/químicaRESUMEN
Phytochromes are bilin-containing photoreceptors that are typically sensitive to the red/far-red region of the visible spectrum. Recently, phytochromes from certain eukaryotic algae have become attractive targets for optogenetic applications because of their unique ability to respond to multiple wavelengths of light. Herein, a combination of time-resolved spectroscopy and structural approaches across picosecond to second timescales have been used to map photochemical mechanisms and structural changes in this atypical group of phytochromes. The photochemistry of an orange/far-red light-sensitive algal phytochrome from Dolihomastix tenuilepis has been investigated by using a combination of visible, IR and X-ray scattering probes. The entire photocycle, correlated with accompanying structural changes in the cofactor/protein, are reported. This study identifies a complex photocycle for this atypical phytochrome. It also highlights a need to combine outcomes from a range of biophysical approaches to unravel complex photochemical and macromolecular processes in multi-domain photoreceptor proteins that are the basis of biological light-mediated signalling.
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Chlorophyta/química , Fitocromo/química , Procesos Fotoquímicos , Conformación Proteica , Espectrofotometría Infrarroja , Difracción de Rayos XRESUMEN
A new series of 3-hydroxy-2-pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16-18) were determined to be greater than that of 2-PAM and comparable to that of HI-6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP-inhibited enzyme have been clarified by molecular-modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE. Blood-brain barrier penetration was predicted for oximes 15-18 based on their physicochemical properties and an in vitro brain membrane permeation assay. Among the evaluated compounds, two morpholine-3-hydroxypyridine aldoxime conjugates proved to be promising reactivators of OP-inhibited cholinesterases. Moreover, efficient ex vivo reactivation of phosphylated native cholinesterases by selected oximes enabled significant hydrolysis of VX, sarin, paraoxon, and cyclosarin in whole human blood, which indicates that the oximes have scavenging potential.
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Barrera Hematoencefálica/metabolismo , Butirilcolinesterasa/metabolismo , Organofosfatos/química , Oximas/química , Barrera Hematoencefálica/química , Butirilcolinesterasa/química , Humanos , Relación Estructura-ActividadRESUMEN
Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1.
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GTP Fosfohidrolasas/metabolismo , Nucleótidos de Guanina/metabolismo , Proteínas/metabolismo , Enfermedades de la Médula Ósea/genética , Enfermedades de la Médula Ósea/metabolismo , Insuficiencia Pancreática Exocrina/genética , Insuficiencia Pancreática Exocrina/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Cinética , Lipomatosis/genética , Lipomatosis/metabolismo , Mutación , Factores de Elongación de Péptidos , Unión Proteica , Proteínas/genética , Ribonucleoproteína Nuclear Pequeña U5 , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Síndrome de Shwachman-DiamondRESUMEN
Darobactins represent a class of ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics featuring a rare bicyclic structure. They target the Bam-complex of Gram-negative bacteria and exhibit in vivo activity against drug-resistant pathogens. First isolated from Photorhabdus species, the corresponding biosynthetic gene clusters (BGCs) are widespread among γ-proteobacteria, including the genera Vibrio, Yersinia, and Pseudoalteromonas (P.). While the organization of the BGC core is highly conserved, a small subset of Pseudoalteromonas carries an extended BGC with additional genes. Here, we report the identification of brominated and dehydrated darobactin derivatives from P. luteoviolacea strains. The marine derivatives are active against multidrug-resistant (MDR) Gram-negative bacteria and showed solubility and plasma protein binding ability different from darobactin A, rendering it more active than darobactin A. The halogenation reaction is catalyzed by DarH, a new class of flavin-dependent halogenases with a novel fold.
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Fenilpropionatos , Fenilpropionatos/metabolismo , Bacterias Gramnegativas/genética , MetabolomaRESUMEN
X-ray radiation induces two main effects at metal centres contained in protein crystals: radiation-induced reduction and radiolysis and a resulting decrease in metal occupancy. In blue multicopper oxidases (BMCOs), the geometry of the active centres and the metal-to-ligand distances change depending on the oxidation states of the Cu atoms, suggesting that these alterations are catalytically relevant to the binding, activation and reduction of O(2). In this work, the X-ray-determined three-dimensional structure of laccase from the basidiomycete Coriolopsis gallica (Cg L), a high catalytic potential BMCO, is described. By combining spectroscopic techniques (UV-Vis, EPR and XAS) and X-ray crystallography, structural changes at and around the active copper centres were related to pH and absorbed X-ray dose (energy deposited per unit mass). Depletion of two of the four active Cu atoms as well as low occupancies of the remaining Cu atoms, together with different conformations of the metal centres, were observed at both acidic pH and high absorbed dose, correlating with more reduced states of the active coppers. These observations provide additional evidence to support the role of flexibility of copper sites during O(2) reduction. This study supports previous observations indicating that interpretations regarding redox state and metal coordination need to take radiation effects explicitly into account.
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Basidiomycota/enzimología , Dominio Catalítico/efectos de la radiación , Cobre/química , Cristalografía por Rayos X , Lacasa/química , Cristalización , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Oxidación-Reducción , Oxígeno/química , Conformación Proteica/efectos de la radiación , Espectrofotometría Ultravioleta , Rayos XRESUMEN
Proliferating cell nuclear antigen (PCNA), a member of the sliding clamp family of proteins, interacts specifically with DNA replication and repair proteins through a small peptide motif called the PCNA-interacting protein or PIP box. PCNA is recognized as one of the key proteins involved in DNA metabolism. In the present study, the recombinant PCNA from Litopenaeus vannamei (LvPCNA) was heterologously overexpressed and purified using metal ion-affinity chromatography. Crystals suitable for diffraction grew overnight using the hanging-drop vapour-diffusion method. LvPCNA crystals belong to space group C2 with unit-cell parameters a=144.6, b=83.4, c=74.3â Å, ß=117.6°. One data set was processed to 3â Å resolution, with an overall Rmeas of 0.09 and a completeness of 93.3%. Initial phases were obtained by molecular replacement using a homology model of LvPCNA as the search model. Refinement and structural analysis are underway. This report is the first successful crystallographic analysis of a marine crustacean decapod shrimp (L. vannamei) proliferating cell nuclear antigen.
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Proteínas de Artrópodos/química , Penaeidae , Antígeno Nuclear de Célula en Proliferación/química , Animales , Proteínas de Artrópodos/biosíntesis , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli , Antígeno Nuclear de Célula en Proliferación/biosíntesisRESUMEN
The rate of radiation damage to macromolecular crystals at both room temperature and 100 K has previously been shown to be reduced by the use of certain radical scavengers. Here the effects of sodium nitrate, an electron scavenger, are investigated at 100 K. For sodium nitrate at a concentration of 0.5 M in chicken egg-white lysozyme crystals, the dose tolerance is increased by a factor of two as judged from the global damage parameters, and no specific structural damage to the disulfide bonds is seen until the dose is greatly in excess (more than a factor of five) of the value at which damage appears in electron density maps derived from a scavenger-free crystal. In the electron density maps, ordered nitrate ions adjacent to the disulfide bonds are seen to lose an O atom, and appear to protect the disulfide bonds. In addition, results reinforcing previous reports on the effectiveness of ascorbate are presented. The mechanisms of action of both scavengers in the crystalline environment are elucidated.
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Frío , Proteínas/química , Cristalización , Espectrofotometría UltravioletaRESUMEN
Membrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Structural information on how proteins form MCS is scarce. We designed an in vitro MCS with two membranes and a pair of tethering proteins suitable for cryo-tomography analysis. It includes VAP-A, an ER transmembrane protein interacting with a myriad of cytosolic proteins, and oxysterol-binding protein (OSBP), a lipid transfer protein that transports cholesterol from the ER to the trans Golgi network. We show that VAP-A is a highly flexible protein, allowing formation of MCS of variable intermembrane distance. The tethering part of OSBP contains a central, dimeric, and helical T-shape region. We propose that the molecular flexibility of VAP-A enables the recruitment of partners of different sizes within MCS of adjustable thickness, whereas the T geometry of the OSBP dimer facilitates the movement of the two lipid-transfer domains between membranes.
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Acetylcholinesterase (AChE) inhibitors (AChEIs) still remain the leading therapeutic options for the symptomatic treatment of cognitive deficits associated with mild-to-moderate Alzheimer's disease. The search for new AChEIs benefits from well-established knowledge of the molecular interactions of selective AChEIs, such as donepezil and related dual binding site inhibitors. Starting from a previously disclosed coumarin-based inhibitor (±)-cis-1, active as racemate in the nanomolar range toward AChE, we proceeded on a double track by (i) achieving chiral resolution of the enantiomers of 1 by HPLC and (ii) preparing two close achiral analogues of 1, i.e., compounds 4 and 6. An eudismic ratio as high as 20 was observed for the (-) enantiomer of cis-1. The X-ray crystal structure of the complex between the (-)-cis-1 eutomer (coded as MC1420) and T. californica AChE was determined at 2.8 Å, and docking calculation results suggested that the eutomer in (1R,3S) absolute configuration should be energetically more favored in binding the enzyme than the eutomer in (1S,3R) configuration. The achiral analogues 4 and 6 were less effective in inhibiting AChE compared to (±)-cis-1, but interestingly butylamide 4 emerged as a potent inhibitor of butyrylcholinesterase (BChE).
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Endosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes with negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and how ESCRT-III shapes membranes is yet unclear. Human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron tomography and AFM. We show that CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes. Combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes and reshape vesicles into helical "corkscrew-like" membrane tubes. Sub-tomogram averaging reveals that the ESCRT-III filaments assemble parallel and locally perpendicular to the tube axis, highlighting the mechanical stresses imposed by ESCRT-III. Our results underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature required for cellular membrane remodeling processes.
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Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Membranas Artificiales , Estrés Mecánico , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Fenómenos Bioquímicos , Microscopía por Crioelectrón , Humanos , Nanotubos , Polimerizacion , Unión Proteica/fisiología , Multimerización de Proteína , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the µs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.
RESUMEN
Phytochromes are photoreceptor proteins that transmit a light signal from a photosensory region to an output domain. Photoconversion involves protein conformational changes whose nature is not fully understood. Here, we use time-resolved X-ray scattering and optical spectroscopy to study the kinetics of structural changes in a full-length cyanobacterial phytochrome and in a truncated form with no output domain. X-ray and spectroscopic signals on the µs/ms timescale are largely independent of the presence of the output domain. On longer time-scales, large differences between the full-length and truncated proteins indicate the timeframe during which the structural transition is transmitted from the photosensory region to the output domain and represent a large quaternary motion. The suggested independence of the photosensory-region dynamics on the µs/ms timescale defines a time window in which the photoreaction can be characterized (e.g. for optogenetic design) independently of the nature of the engineered output domain.
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Proteínas Bacterianas/química , Proteínas Bacterianas/efectos de la radiación , Luz , Fitocromo/química , Fitocromo/efectos de la radiación , Proteínas Quinasas/química , Proteínas Quinasas/efectos de la radiación , Dispersión de Radiación , Synechocystis/química , Deinococcus/química , Cinética , Modelos Moleculares , Fotorreceptores Microbianos , Conformación Proteica/efectos de la radiación , Transducción de Señal/efectos de la radiación , Espectroscopía de Absorción de Rayos X , Rayos XRESUMEN
Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE. X-ray structures and molecular docking indicate that structural modification of the tetrahydroacridine might decrease inhibition without affecting reactivation. The chlorinated derivative was synthesized and, in line with the prediction, displayed a 10-fold decrease in inhibition but no significant decrease in reactivation efficiency. X-ray structures with the derivative rationalize this outcome. We thus show that rational design based on structural studies permits the refinement of new-generation pyridine aldoxime reactivators that may be more effective in the treatment of nerve agent intoxication.
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Reactivadores de la Colinesterasa/química , Reactivadores de la Colinesterasa/farmacología , Agentes Nerviosos/toxicidad , Relación Estructura-Actividad , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Animales , Dominio Catalítico , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos/métodos , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Agentes Nerviosos/química , Cloruro de Obidoxima/farmacología , Compuestos Organofosforados/química , Compuestos Organofosforados/toxicidadRESUMEN
Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecond timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.
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Structure-based virtual screening of two libraries containing 567â¯981 molecules was used to discover novel, selective BuChE inhibitors, which are potentially superior symptomatic treatments in late-stage Alzheimer's disease. Compound 16 was identified as a highly selective submicromolar inhibitor of BuChE (huBuChE IC50 = 0.443 µM) with high permeability in the PAMPA-BBB model. The X-ray crystal structure of huBuChE in complex with 16 revealed the atomic-level interactions and offers opportunities for further development of the series.