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1.
Nature ; 615(7954): 939-944, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36949205

RESUMO

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.


Assuntos
Rodopsina , Visão Ocular , Animais , Sítios de Ligação/efeitos da radiação , Cristalografia , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Isomerismo , Fótons , Ligação Proteica/efeitos da radiação , Conformação Proteica/efeitos da radiação , Retinaldeído/química , Retinaldeído/metabolismo , Retinaldeído/efeitos da radiação , Rodopsina/química , Rodopsina/metabolismo , Rodopsina/efeitos da radiação , Fatores de Tempo , Visão Ocular/fisiologia , Visão Ocular/efeitos da radiação
2.
Nature ; 583(7815): 314-318, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32499654

RESUMO

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.


Assuntos
Flavobacteriaceae/química , Rodopsinas Microbianas/química , Rodopsinas Microbianas/efeitos da radiação , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/efeitos da radiação , Sítios de Ligação , Cristalografia , Elétrons , Transporte de Íons , Isomerismo , Lasers , Prótons , Teoria Quântica , Retinaldeído/química , Retinaldeído/metabolismo , Bases de Schiff/química , Sódio/metabolismo , Análise Espectral , Eletricidade Estática , Fatores de Tempo
3.
Angew Chem Int Ed Engl ; 59(48): 21656-21662, 2020 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-32780931

RESUMO

Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxide to form the catalytic intermediate compound I (FeIV =O and a porphyrin cation radical). Using serial femtosecond X-ray crystallography (SFX), we have determined the pristine structures of the FeIII and FeIV =O redox states of a B-type DyP. These structures reveal a water-free distal heme site that, together with the presence of an asparagine, imply the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.


Assuntos
Arginina/metabolismo , Corantes/metabolismo , Heme/metabolismo , Compostos de Ferro/metabolismo , Oxigênio/metabolismo , Peroxidase/metabolismo , Arginina/química , Biocatálise , Corantes/química , Cristalografia por Raios X , Heme/química , Compostos de Ferro/química , Modelos Moleculares , Oxirredução , Oxigênio/química , Peroxidase/química , Streptomyces lividans/enzimologia
4.
Appl Opt ; 57(30): 9032-9039, 2018 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-30461891

RESUMO

Macromolecular crystallography often requires focused high-intensity x-ray beams for solving challenging protein structures from micrometer-sized crystals using current synchrotron radiation sources. The design of optical focusing schemes for hard x-rays showing high efficiency and flexibility in beam size is therefore continuously pursued. Here, we present an innovative solution based on a two-stage demagnification of the undulator source for photon energies from 6 keV to 19 keV, commissioned at the X10SA beamline of the Swiss Light Source, where a secondary source is imaged by two crossed silicon kinoform x-ray diffractive lenses with 75 nm outermost zone width. A source-size limited spot with a size of 4.8 µm×1.7 µm(h×v,FWHM) and flux of 7.5×1010 photons/s at 12.4 keV is demonstrated at the sample position.

5.
Adv Exp Med Biol ; 922: 137-149, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27553240

RESUMO

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a powerful method to determine high-resolution structures of pharmaceutically relevant membrane proteins. Recently, the technology has been adapted to carry out serial millisecond crystallography (SMX) at synchrotron sources, where beamtime is more abundant. In an injector-based approach, crystals grown in lipidic cubic phase (LCP) or embedded in viscous medium are delivered directly into the unattenuated beam of a microfocus beamline. Pilot experiments show the application of microjet-based SMX for solving the structure of a membrane protein and compatibility of the method with de novo phasing. Planned synchrotron upgrades, faster detectors and software developments will go hand-in-hand with developments at free-electron lasers to provide a powerful methodology for solving structures from microcrystals at room temperature, ligand screening or crystal optimization for time-resolved studies with minimal or no radiation damage.


Assuntos
Cristalografia por Raios X/métodos , Proteínas de Membrana/química , Cristalização , Cristalografia por Raios X/instrumentação , Elétrons , Previsões , Humanos , Lasers , Bicamadas Lipídicas , Proteínas de Membrana/efeitos da radiação , Síncrotrons , Temperatura , Fatores de Tempo , Viscosidade
6.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 1): 27-35, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25615857

RESUMO

Combining macromolecular crystallography with in crystallo micro-spectrophotometry yields valuable complementary information on the sample, including the redox states of metal cofactors, the identification of bound ligands and the onset and strength of undesired photochemistry, also known as radiation damage. However, the analysis and processing of the resulting data differs significantly from the approaches used for solution spectrophotometric data. The varying size and shape of the sample, together with the suboptimal sample environment, the lack of proper reference signals and the general influence of the X-ray beam on the sample have to be considered and carefully corrected for. In the present article, how to characterize and treat these sample-dependent artefacts in a reproducible manner is discussed and the SLS-APE in situ, in crystallo optical spectroscopy data-analysis toolbox is demonstrated.


Assuntos
Cristalografia/métodos , Espectrofotometria Ultravioleta/métodos , Análise Espectral Raman/métodos , Automação , Fluorescência
7.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 2): 387-97, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25664750

RESUMO

Recent advances in synchrotron sources, beamline optics and detectors are driving a renaissance in room-temperature data collection. The underlying impetus is the recognition that conformational differences are observed in functionally important regions of structures determined using crystals kept at ambient as opposed to cryogenic temperature during data collection. In addition, room-temperature measurements enable time-resolved studies and eliminate the need to find suitable cryoprotectants. Since radiation damage limits the high-resolution data that can be obtained from a single crystal, especially at room temperature, data are typically collected in a serial fashion using a number of crystals to spread the total dose over the entire ensemble. Several approaches have been developed over the years to efficiently exchange crystals for room-temperature data collection. These include in situ collection in trays, chips and capillary mounts. Here, the use of a slowly flowing microscopic stream for crystal delivery is demonstrated, resulting in extremely high-throughput delivery of crystals into the X-ray beam. This free-stream technology, which was originally developed for serial femtosecond crystallography at X-ray free-electron lasers, is here adapted to serial crystallography at synchrotrons. By embedding the crystals in a high-viscosity carrier stream, high-resolution room-temperature studies can be conducted at atmospheric pressure using the unattenuated X-ray beam, thus permitting the analysis of small or weakly scattering crystals. The high-viscosity extrusion injector is described, as is its use to collect high-resolution serial data from native and heavy-atom-derivatized lysozyme crystals at the Swiss Light Source using less than half a milligram of protein crystals. The room-temperature serial data allow de novo structure determination. The crystal size used in this proof-of-principle experiment was dictated by the available flux density. However, upcoming developments in beamline optics, detectors and synchrotron sources will enable the use of true microcrystals. This high-throughput, high-dose-rate methodology provides a new route to investigating the structure and dynamics of macromolecules at ambient temperature.


Assuntos
Cristalografia por Raios X/instrumentação , Síncrotrons/instrumentação , Animais , Galinhas , Cristalografia por Raios X/economia , Cristalografia por Raios X/métodos , Desenho de Equipamento , Modelos Moleculares , Muramidase/química , Temperatura , Viscosidade
8.
J Biol Inorg Chem ; 20(6): 949-56, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26100643

RESUMO

Cytochromes c', that occur in methanotrophic, denitrifying and photosynthetic bacteria, form unusual proximal penta-coordinate NO complexes via a hexa-coordinate distal NO intermediate. Their NO binding properties are similar to those of the eukaryotic NO sensor, soluble guanylate cyclase, for which they provide a valuable structural model. Previous studies suggested that hydrogen bonding between the displaced proximal histidine (His120) ligand (following its dissociation from heme due to trans effects from the distally bound NO) and a conserved aspartate residue (Asp121) could play a key role in allowing proximal NO binding to occur. We have characterized three variants of Alcaligenes xylosoxidans cytochrome c' (AXCP) where Asp121 has been replaced by Ala, Ile and Gln, respectively. In all variants, hydrogen bonding between residue 121 and His120 is abolished yet 5-coordinate proximal NO species are still formed. Our data therefore demonstrate that the His120-Asp121 bond is not essential for proximal NO binding although it likely provides an energy minimum for the displaced His ligand. All variants have altered proximal pocket structure relative to native AXCP.


Assuntos
Citocromos c'/química , Histidina/química , Óxido Nítrico/química , Achromobacter denitrificans , Citocromos c'/ultraestrutura , Ligação de Hidrogênio , Ligantes , Modelos Moleculares
9.
Proteins ; 82(10): 2831-41, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25044250

RESUMO

Serine hydroxymethyltransferase (SHMT) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme belonging to the fold type I superfamily, which catalyzes in vivo the reversible conversion of l-serine and tetrahydropteroylglutamate (H4PteGlu) to glycine and 5,10-methylenetetrahydropteroylglutamate (5,10-CH2-H4PteGlu). The SHMT from the psychrophilic bacterium Psychromonas ingrahamii (piSHMT) had been recently purified and characterized. This enzyme was shown to display catalytic and stability properties typical of psychrophilic enzymes, namely high catalytic activity at low temperature and thermolability. To gain deeper insights into the structure-function relationship of piSHMT, the three-dimensional structure of its apo form was determined by X-ray crystallography. Homology modeling techniques were applied to build a model of the piSHMT holo form. Comparison of the two forms unraveled the conformation modifications that take place when the apo enzyme binds its cofactor. Our results show that the apo form is in an "open" conformation and possesses four (or five, in chain A) disordered loops whose electron density is not visible by X-ray crystallography. These loops contain residues that interact with the PLP cofactor and three of them are localized in the major domain that, along with the small domain, constitutes the single subunit of the SHMT homodimer. Cofactor binding triggers a rearrangement of the small domain that moves toward the large domain and screens the PLP binding site at the solvent side. Comparison to the mesophilic apo SHMT from Salmonella typhimurium suggests that the backbone conformational changes are wider in psychrophilic SHMT.


Assuntos
Proteínas de Bactérias/química , Coenzimas/metabolismo , Gammaproteobacteria/enzimologia , Glicina Hidroximetiltransferase/química , Modelos Moleculares , Fosfato de Piridoxal/metabolismo , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Coenzimas/química , Cristalografia por Raios X , Bases de Dados de Proteínas , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Gammaproteobacteria/crescimento & desenvolvimento , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Fosfato de Piridoxal/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/enzimologia , Salmonella typhimurium/crescimento & desenvolvimento
10.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 5): 1289-96, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24816098

RESUMO

It is crucial to assign the correct redox and ligand states to crystal structures of proteins with an active redox centre to gain valid functional information and prevent the misinterpretation of structures. Single-crystal spectroscopies, particularly when applied in situ at macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. Single-crystal resonance Raman spectroscopy was carried out in combination with macromolecular crystallography on Swiss Light Source beamline X10SA using cytochrome c' from Alcaligenes xylosoxidans. This allowed the fingerprinting and validation of different redox and ligand states, identification of vibrational modes and identification of intermediates together with monitoring of radiation-induced changes. This combined approach provides a powerful tool to obtain complementary data and correctly assign the true oxidation and ligand state(s) in redox-protein crystals.


Assuntos
Cristalografia por Raios X/métodos , Citocromos c/química , Hemeproteínas/química , Análise Espectral Raman , Alcaligenes/química , Citocromos c/metabolismo , Hemeproteínas/metabolismo , Ligantes , Modelos Moleculares , Oxirredução , Conformação Proteica
11.
J Am Chem Soc ; 136(1): 137-46, 2014 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-24328211

RESUMO

Cellular retinaldehyde-binding protein (CRALBP) chaperones 11-cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/vis and (1)H NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal. The X-ray structure of the CRALBP mutant R234W:9-cis-retinal complex at 1.9 Å resolution revealed a niche in the binding pocket for 9-cis-aldehyde different from that reported for 11-cis-retinal. Combined computational, kinetic, and structural data lead us to propose an isomerization mechanism catalyzed by a network of buried waters. Our findings highlight a specific role of water molecules in both CRALBP-assisted specificity toward 9-cis-retinal and its thermal isomerase activity yielding 9,13-dicis-retinal. Kinetic data from two point mutants of CRALBP support an essential role of Glu202 as the initial proton donor in this isomerization reaction.


Assuntos
Proteínas de Transporte/metabolismo , Isomerases/química , Isomerases/metabolismo , Retinaldeído/química , Proteínas de Transporte/química , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Diterpenos , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Teoria Quântica , Especificidade por Substrato
12.
J Synchrotron Radiat ; 21(Pt 2): 340-51, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24562555

RESUMO

A new diffractometer for microcrystallography has been developed for the three macromolecular crystallography beamlines of the Swiss Light Source. Building upon and critically extending previous developments realised for the high-resolution endstations of the two undulator beamlines X06SA and X10SA, as well as the super-bend dipole beamline X06DA, the new diffractometer was designed to the following core design goals. (i) Redesign of the goniometer to a sub-micrometer peak-to-peak cylinder of confusion for the horizontal single axis. Crystal sizes down to at least 5 µm and advanced sample-rastering and scanning modes are supported. In addition, it can accommodate the new multi-axis goniometer PRIGo (Parallel Robotics Inspired Goniometer). (ii) A rapid-change beam-shaping element system with aperture sizes down to a minimum of 10 µm for microcrystallography measurements. (iii) Integration of the on-axis microspectrophotometer MS3 for microscopic sample imaging with 1 µm image resolution. Its multi-mode optical spectroscopy module is always online and supports in situ UV/Vis absorption, fluorescence and Raman spectroscopy. (iv) High stability of the sample environment by a mineral cast support construction and by close containment of the cryo-stream. Further features are the support for in situ crystallization plate screening and a minimal achievable detector distance of 120 mm for the Pilatus 6M, 2M and the macromolecular crystallography group's planned future area detector Eiger 16M.

13.
Nat Chem ; 16(4): 624-632, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38225270

RESUMO

Charge-transfer reactions in proteins are important for life, such as in photolyases which repair DNA, but the role of structural dynamics remains unclear. Here, using femtosecond X-ray crystallography, we report the structural changes that take place while electrons transfer along a chain of four conserved tryptophans in the Drosophila melanogaster (6-4) photolyase. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at a key asparagine, at a conserved salt bridge, and by rearrangements of nearby water molecules. We detect charge-induced structural changes close to the second tryptophan from 1 ps to 20 ps, identifying a nearby methionine as an active participant in the redox chain, and from 20 ps around the fourth tryptophan. The photolyase undergoes highly directed and carefully timed adaptations of its structure. This questions the validity of the linear solvent response approximation in Marcus theory and indicates that evolution has optimized fast protein fluctuations for optimal charge transfer.


Assuntos
Desoxirribodipirimidina Fotoliase , Humanos , Animais , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Triptofano/química , Elétrons , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Transporte de Elétrons , Cristalografia por Raios X
14.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 1): 137-40, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23275172

RESUMO

Despite their high physiological relevance, haemoglobin crystal structures with NO bound to haem constitute less than 1% of the total ligated haemoglobins (Hbs) deposited in the Protein Data Bank. The major difficulty in obtaining NO-ligated Hbs is most likely to be related to the oxidative denitrosylation caused by the high reactivity of the nitrosylated species with O(2). Here, using Raman-assisted X-ray crystallography, it is shown that under X-ray exposure (at four different radiation doses) crystals of nitrosylated haemoglobin from Trematomus bernacchii undergo a transition, mainly in the ß chains, that generates a pentacoordinate species owing to photodissociation of the Fe-NO bond. These data provide a physical explanation for the low number of nitrosylated Hb structures available in the literature.


Assuntos
Hemoglobinas/química , Hemoglobinas/efeitos da radiação , Óxido Nítrico/química , Óxido Nítrico/efeitos da radiação , Processos Fotoquímicos , Análise Espectral Raman , Animais , Cristalografia por Raios X/métodos , Peixes , Hemoglobinas/metabolismo , Microespectrofotometria/métodos , Óxido Nítrico/metabolismo , Processos Fotoquímicos/efeitos da radiação , Análise Espectral Raman/métodos
15.
J Synchrotron Radiat ; 20(Pt 5): 765-76, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23955041

RESUMO

The combination of X-ray diffraction experiments with optical methods such as Raman, UV/Vis absorption and fluorescence spectroscopy greatly enhances and complements the specificity of the obtained information. The upgraded version of the in situ on-axis micro-spectrophotometer, MS2, at the macromolecular crystallography beamline X10SA of the Swiss Light Source is presented. The instrument newly supports Raman and resonance Raman spectroscopy, in addition to the previously available UV/Vis absorption and fluorescence modes. With the recent upgrades of the spectral bandwidth, instrument stability, detection efficiency and control software, the application range of the instrument and its ease of operation were greatly improved. Its on-axis geometry with collinear X-ray and optical axes to ensure optimal control of the overlap of sample volumes probed by each technique is still unique amongst comparable facilities worldwide and the instrument has now been in general user operation for over two years.


Assuntos
Cristalografia por Raios X , Análise Espectral Raman/instrumentação , Desenho Assistido por Computador , Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Desenho de Equipamento , Substâncias Macromoleculares , Reprodutibilidade dos Testes , Software , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Espectrofotometria Ultravioleta/instrumentação , Espectrofotometria Ultravioleta/métodos , Análise Espectral Raman/métodos
16.
IUCrJ ; 10(Pt 6): 729-737, 2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37830774

RESUMO

Serial and time-resolved macromolecular crystallography are on the rise. However, beam time at X-ray free-electron lasers is limited and most third-generation synchrotron-based macromolecular crystallography beamlines do not offer the necessary infrastructure yet. Here, a new setup is demonstrated, based on the JUNGFRAU detector and Jungfraujoch data-acquisition system, that enables collection of kilohertz serial crystallography data at fourth-generation synchrotrons. More importantly, it is shown that this setup is capable of collecting multiple-time-point time-resolved protein dynamics at kilohertz rates, allowing the probing of microsecond to second dynamics at synchrotrons in a fraction of the time needed previously. A high-quality complete X-ray dataset was obtained within 1 min from lysozyme microcrystals, and the dynamics of the light-driven sodium-pump membrane protein KR2 with a time resolution of 1 ms could be demonstrated. To make the setup more accessible for researchers, downstream data handling and analysis will be automated to allow on-the-fly spot finding and indexing, as well as data processing.

17.
Nat Commun ; 14(1): 903, 2023 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-36807348

RESUMO

The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we capture the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.


Assuntos
Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Cristalografia , Ligantes , Microtúbulos/metabolismo , Cristalografia por Raios X
18.
Nat Commun ; 14(1): 7956, 2023 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-38042952

RESUMO

Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.


Assuntos
Elétrons , Proteínas , Cristalografia , Cristalografia por Raios X , Proteínas/química , Síncrotrons , Lasers
19.
Science ; 382(6674): 1015-1020, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-38033070

RESUMO

Photolyase is an enzyme that uses light to catalyze DNA repair. To capture the reaction intermediates involved in the enzyme's catalytic cycle, we conducted a time-resolved crystallography experiment. We found that photolyase traps the excited state of the active cofactor, flavin adenine dinucleotide (FAD), in a highly bent geometry. This excited state performs electron transfer to damaged DNA, inducing repair. We show that the repair reaction, which involves the lysis of two covalent bonds, occurs through a single-bond intermediate. The transformation of the substrate into product crowds the active site and disrupts hydrogen bonds with the enzyme, resulting in stepwise product release, with the 3' thymine ejected first, followed by the 5' base.


Assuntos
Desoxirribodipirimidina Fotoliase , Cristalografia , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/metabolismo , Reparo do DNA , Dano ao DNA , Transporte de Elétrons
20.
Chem Commun (Camb) ; 58(31): 4889-4892, 2022 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-35352724

RESUMO

We present the structure of a photoactivated animal (6-4) photolyase in its radical pair state, captured by serial crystallography. We observe how a conserved asparigine moves towards the semiquinone FAD chromophore and stabilizes it by hydrogen bonding. Several amino acids around the final tryptophan radical rearrange, opening it up to the solvent. The structure explains how the protein environment stabilizes the radical pair state, which is crucial for function of (6-4) photolyases and cryptochromes.


Assuntos
Desoxirribodipirimidina Fotoliase , Aminoácidos , Animais , Criptocromos/química , Criptocromos/metabolismo , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/metabolismo , Flavina-Adenina Dinucleotídeo/química , Triptofano/química
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