RESUMO
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a â¼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.
Assuntos
Arrestina/química , Arrestina/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Lasers , Camundongos , Modelos Moleculares , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Transdução de Sinais , Raios XRESUMO
Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.
Assuntos
Cristalografia por Raios X , Cianobactérias/química , Modelos Moleculares , Complexo de Proteína do Fotossistema II/química , Estrutura Terciária de ProteínaRESUMO
To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 µm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 µm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.
Assuntos
Cristalografia/métodos , Elétrons , Granulovirus/ultraestrutura , Peptídeos e Proteínas de Sinalização Intercelular/química , Lasers , Cristalografia/instrumentação , Granulovirus/química , Modelos Moleculares , Progranulinas , Estrutura Secundária de Proteína , SíncrotronsRESUMO
We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.
Assuntos
Transferência de Energia/efeitos da radiação , Lasers , Ficobiliproteínas/efeitos da radiação , Ficobiliproteínas/ultraestrutura , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos , Ficobiliproteínas/química , Conformação Proteica/efeitos da radiação , Doses de RadiaçãoRESUMO
The powerful and specific molecular-recognition system present in the base-pairing of DNA allows for the design of a plethora of nanostructures. In this work, the crystallization of a self-assembling three-dimensional B-DNA nanostructure is described. The DNA nanostructure consists of six single-stranded oligonucleotides that hybridize to form a three-dimensional tetrahedron of 80 kDa in molecular mass and 20 bp on each edge. Crystals of the tetrahedron have been successfully produced and characterized. These crystals may form the basis for an X-ray structure of the tetrahedron in the future. Nucleotide crystallography poses many challenges, leading to the fact that only 1352 X-ray structures of nucleic acids have been solved compared with more than 80,000 protein structures. In this work, the crystallization optimization for three-dimensional tetrahedra is also described, with the eventual goal of producing nanocrystals to overcome the radiation-damage obstacle by the use of free-electron laser technology in the future.
Assuntos
DNA/química , Nanoestruturas/química , Conformação de Ácido Nucleico , Cristalização , Cristalografia por Raios XRESUMO
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
Assuntos
Cristalografia por Raios X/métodos , Ferredoxinas/ultraestrutura , Lasers , Nanoestruturas/ultraestrutura , Difração de Raios X/métodos , Elétrons , Conformação Proteica , Raios XRESUMO
OBJECTIVE: In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal transport and barrier function. The mechanisms of cytokine action have usually been studied in cell culture models only; therefore the aim of this study was to establish an in vitro model based on native intestine to analyze distinct cytokine effects on barrier function, mucosal structure, and inherent regulatory mechanisms. MATERIAL AND METHODS: Rat colon was exposed to tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) in Ussing chambers. Transepithelial resistance (R(t)) and (3)H-mannitol fluxes were measured for characterization of the paracellular pathway. Transcellular transport was analyzed by horseradish peroxidase (HRP) flux measurements. Expression and distribution of tight junction proteins were characterized in immunoblots and by means of confocal laser-scanning microscopy (LSM). RESULTS: Colonic viability could be preserved for 20 h in a specialized in vitro set-up. This was sufficient to alter mucosal architecture with crypt surface reduction. R(t) was decreased (101+/-10 versus 189+/-10 Omega x cm(2)) with a parallel increase in mannitol permeability after cytokine exposure. Tight junction proteins claudin-1, -5, -7, and occludin decreased (45+/-10%, 16+/-7%, 42+/-8%, and 42+/-13% of controls, respectively), while claudin-2 increased to 208+/-32%. Occludin and claudin-1 translocated from the plasma membrane to the cytoplasm. HRP flux increased from 0.73+/-0.09 to 8.55+/-2.92 pmol x h(-1) x cm(-2). CONCLUSIONS: A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFalpha and IFNgamma alters mucosal morphology and impairs epithelial barrier function by up-regulation of the paracellular pore-former claudin-2 and down-regulation of the barrier-builders claudin-1, -5, and -7. These alterations resemble changes seen in IBD and thus underline their prominent role in IBD pathogenicity.
Assuntos
Doenças Inflamatórias Intestinais/fisiopatologia , Interferon gama/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiopatologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Modelos Animais de Doenças , Técnicas In Vitro , Doenças Inflamatórias Intestinais/imunologia , Masculino , Ratos , Ratos WistarRESUMO
The H+-ATP synthase from chloroplasts, CF0F1, was isolated, reconstituted into liposomes and ATP synthesis activity was measured after energization of the proteoliposomes with an acid-base transition. The ATP yield was measured as a function of the reaction time after energization, the data were fitted by an exponential function and the initial rate was calculated from the fit parameters. CF0F1 was reconstituted by detergent dialysis in asolectin liposomes and phosphatidylcholine/phosphatidic acid (PtdCho/PtdAc from egg yolk) liposomes. In asolectin liposomes, high initial rates of ATP synthesis (up to 400 s(-1)) were observed with a rapid decline of the rate; in PtdCho/PtdAc liposomes the initial rate is smaller (up to 200 s(-1)), but the decline of the activity is slower. CF0F1 was reconstituted into PtdCho/PtdAc liposomes either by detergent dialysis or into reverse phase liposomes. The dependence of the rate of ATP synthesis on the phosphate concentration was measured with both types of proteoliposomes. The data can be described by Michaelis-Menten kinetics with a K(M) value of 350 microM for reverse phase liposomes and a K(M) value of 970 microM for dialysis liposomes. Both K(M) values depend neither on the magnitude of DeltapH nor on the electric potential difference, whereas V(max) decreases strongly with decreasing energization. At low phosphate concentration, there are small deviations from Michaelis-Menten kinetics. The measured rates are higher than those calculated from the fitted Michaelis-Menten parameters. This effect is interpreted as evidence that more than one phosphate binding site is involved in ATP synthesis.
Assuntos
Trifosfato de Adenosina/biossíntese , ATPases de Cloroplastos Translocadoras de Prótons/isolamento & purificação , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Cloroplastos/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , ATPases de Cloroplastos Translocadoras de Prótons/química , Detergentes/química , Concentração de Íons de Hidrogênio , Lipossomos/síntese química , Lipossomos/química , Lipossomos/metabolismo , Potenciais da Membrana/fisiologia , Oxirredução , Fosfatos/metabolismo , Fosfatidilcolinas , Fosfolipídeos/química , Proteolipídeos/química , Proteolipídeos/metabolismo , Spinacia oleracea/enzimologiaRESUMO
Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
Assuntos
Membrana Celular/enzimologia , Diacilglicerol Quinase/química , Escherichia coli/enzimologia , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Domínio Catalítico , Membrana Celular/química , Cristalografia por Raios X , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Escherichia coli/química , Escherichia coli/genética , Modelos Moleculares , Conformação ProteicaRESUMO
Serial femtosecond crystallography (SFX) is a new emerging method, where X-ray diffraction data are collected from a fully hydrated stream of nano- or microcrystals of biomolecules in their mother liquor using high-energy, X-ray free-electron lasers. The success of SFX experiments strongly depends on the ability to grow large amounts of well-ordered nano/microcrystals of homogeneous size distribution. While methods to grow large single crystals have been extensively explored in the past, method developments to grow nano/microcrystals in sufficient amounts for SFX experiments are still in their infancy. Here, we describe and compare three methods (batch, free interface diffusion (FID) and FID centrifugation) for growth of nano/microcrystals for time-resolved SFX experiments using the large membrane protein complex photosystem II as a model system.
Assuntos
Cristalização/métodos , Cristalografia por Raios X/métodos , Elétrons , Lasers , Complexo de Proteína do Fotossistema II/química , Difração de Raios X/métodos , Cianobactérias , Nanopartículas/química , Conformação ProteicaRESUMO
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3â Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5â Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.
RESUMO
Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-µm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.
Assuntos
Cristalografia/métodos , Lipídeos/química , Proteínas de Membrana/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment.
Assuntos
Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Receptores Acoplados a Proteínas G/química , Humanos , Lasers , Conformação Proteica , Receptor 5-HT2B de Serotonina/química , Receptor 5-HT2B de Serotonina/efeitos da radiação , Receptores Acoplados a Proteínas G/efeitos da radiação , Fatores de TempoRESUMO
Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.
Assuntos
Cristalografia por Raios X/métodos , Hyphomicrobiaceae/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Conformação ProteicaRESUMO
The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.
Assuntos
Catepsina B/química , Proteínas de Protozoários/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Catepsina B/antagonistas & inibidores , Cristalização , Cristalografia por Raios X , Precursores Enzimáticos/química , Glicosilação , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas de Protozoários/antagonistas & inibidores , Células Sf9 , Spodoptera , Raios XRESUMO
Photosynthesis is the major process that converts solar energy into chemical energy on Earth. Two and a half billion years ago, the ancestors of cyanobacteria were able to use water as electron source for the photosynthetic process, thereby evolving oxygen and changing the atmosphere of our planet Earth. Two large membrane protein complexes, Photosystems I and II, catalyze the primary step in this energy conversion, the light-induced charge separation across the photosynthetic membrane. This chapter describes and compares the structure of two Photosystems and discusses their function in respect to the mechanism of light harvesting, electron transfer and water splitting.
Assuntos
Fotossíntese , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema II , Catálise , Cianobactérias/metabolismo , Planeta Terra , Transporte de Elétrons , Proteínas de Membrana/química , Modelos Biológicos , Conformação Molecular , Oxigênio/química , Plantas/metabolismo , Estrutura Terciária de Proteína , Água/química , Raios XRESUMO
Photosystem I is one of the most fascinating membrane protein complexes for which a structure has been determined. It functions as a bio-solar energy converter, catalyzing one of the first steps of oxygenic photosynthesis. It captures the light of the sun by means of a large antenna system, consisting of chlorophylls and carotenoids, and transfers the energy to the center of the complex, driving the transmembrane electron transfer from plastoquinone to ferredoxin. Cyanobacterial Photosystem I is a trimer consisting of 36 proteins to which 381 cofactors are non-covalently attached. This review discusses the complex function of Photosystem I based on the structure of the complex at 2.5 A resolution as well as spectroscopic and biochemical data.