RESUMO
For the two proteins myoglobin and fluoroacetate dehalogenase, we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of micron-sized crystals, the same sample delivery device, and the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins, reasonable data statistics can be obtained with approximately 5000 room-temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that the quality of diffraction data obtained from these samples is linked to the properties of the crystals rather than to the radiation source. Therefore, for other systems with similar properties, time-resolved experiments can be conducted at the radiation source that best matches the desired time resolution.
Assuntos
Proteínas , Síncrotrons , Cristalografia por Raios XRESUMO
We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.
RESUMO
G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange within the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family.
Assuntos
Rodopsina/química , Rodopsina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Citoplasma/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Fótons , Conformação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Doenças Retinianas/metabolismo , Transdução de SinaisRESUMO
To perform their functions within an organism, or to adapt to the environment as single cells, living cells react to signals detected by highly specialized receptor proteins. These include the G-protein coupled receptors (GPCRs), a receptor family, which comprises more than 1000 members, and is of outstanding significance in basic research and medical application. An archetype of a GPCR is the visual pigment rhodopsin, the photoreceptor of the retinal rod cell. Biophysical methods have largely contributed to the elucidation of rhodopsin structure and function, as well as of the corresponding signal cascade. This article discusses some of the more recent developments.
Assuntos
Luz , Transdução de Sinais/fisiologia , Visão Ocular/fisiologia , Animais , Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Rodopsina/química , Rodopsina/fisiologiaRESUMO
The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After light-induced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.
Assuntos
Prótons , Retinaldeído/química , Rodopsina/química , Rodopsina/metabolismo , Transdução de Sinais , Animais , Células COS , Bovinos , Olho/química , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Concentração de Íons de Hidrogênio , Luz , Modelos Biológicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Retinaldeído/análogos & derivados , Bases de Schiff/química , Análise Espectral , Transducina/química , Raios UltravioletaAssuntos
Rodopsina/química , Rodopsina/metabolismo , Transducina/química , Transducina/metabolismo , Biofísica/métodos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Luz , Bicamadas Lipídicas , Fotólise , Espalhamento de Radiação , Espectrometria de Fluorescência/métodos , Espectrofotometria/métodos , Ressonância de Plasmônio de Superfície/métodosRESUMO
The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subunit of G(t), are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the gamma-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G(t) (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the alpha subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the alpha subunit of G(t) and plays a role in the regulation of betagamma subunit binding.
Assuntos
Rodopsina/química , Rodopsina/metabolismo , Transducina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biofísica/métodos , Bovinos , Relação Dose-Resposta a Droga , Ativação Enzimática , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Rodopsina/genética , Fatores de TempoRESUMO
Functional coupling of the human thrombin receptor PAR1 (protease-activated receptor 1) with the retinal rod G-protein transducin (Gt, a member of the Gi family) was studied in a reconstituted system of membranes from Sf9 cells expressing the thrombin receptor and purified Gt from bovine rod outer segments. TRAP6-agonist-activated PAR1 interacts productively with the distant G-protein. Agonist-dependent Gt activation was measured using a real-time fluorimetric GTP[S]-binding assay and membranes from Sf9 cells. To characterize nucleotide-exchange catalysis by PAR1, we analyzed dependence on nucleotides, temperature and pH. Activation was inhibited by low GDP concentrations (IC50 = 5.2 +/- 1.5 microM at 5 microM GTP[S]), indicating that receptor-Gt coupling, followed by instantaneous GDP release, is rate limiting under the conditions (25 degrees C). Arrhenius plots of the temperature dependence reflect an apparent Ea of 60 +/- 3.5 kJ.mol-1. Evaluation of the pH/rate profiles of Gt activation indicates that the activating conformation of the receptor is determined by protonation of a titratable group with an apparent pKa of 6.4. This supports the idea that the active state of agonist-bound PAR1 depends on forced protonation, indicating possible analogies to the scheme established for rhodopsin.
Assuntos
Receptores de Trombina/metabolismo , Transducina/metabolismo , Animais , Bovinos , Linhagem Celular , Expressão Gênica , Nucleotídeos de Guanina/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Receptor PAR-1 , Receptores de Trombina/genética , Rodopsina/metabolismo , SpodopteraRESUMO
G protein-coupled receptors (GPCRs) constitute an abundant family of membrane receptors of high pharmacological interest. Cell-based assays are the predominant means of assessing GPCR activation, but are limited by their inherent complexity. Functional molecular assays that directly and specifically report G protein activation by receptors could offer substantial advantages. We present an approach to immobilize receptors stably and with defined orientation to substrates. By surface plasmon resonance (SPR), we were able to follow ligand binding, G protein activation, and receptor deactivation of a representative GPCR, bovine rhodopsin. Microcontact printing was used to produce micrometer-sized patterns with high contrast in receptor activity. These patterns can be used for local referencing to enhance the sensitivity of chip-based assays. The immobilized receptor was stable both for hours and during several activation cycles. A ligand dose-response curve with the photoactivatable agonist 11-cis-retinal showed a half-maximal signal at 120 nM. Our findings may be useful to develop novel assay formats for GPCRs based on receptor immobilization to solid supports, particularly to sensor surfaces.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/metabolismo , Ressonância de Plasmônio de Superfície , Animais , Biotinilação , Bovinos , Ligantes , Rodopsina/metabolismoRESUMO
Photoactivation of the retinal photoreceptor rhodopsin proceeds through a cascade of intermediates, resulting in protein-protein interactions catalyzing the activation of the G-protein transducin (Gt). Using stabilization and photoregeneration of the receptor's signaling state and Gt activation assays, we provide evidence for a two-site sequential fit mechanism of Gt activation. We show that the C-terminal peptide from the Gt gamma-subunit, Gtgamma(50-71)farnesyl, can replace the holoprotein in stabilizing rhodopsin's active intermediate metarhodopsin II (MII). However, the peptide cannot replace the Gtbeta gamma complex in direct activation assays. Competition by Gtgamma(50-71)farnesyl with Gt for the active receptor suggests a pivotal role for Gtbeta gamma in signal transfer from MII to Gt. MII stabilization and competition is also found for the C-terminal peptide from the Gt alpha-subunit, Gtalpha(340-350), but the capacity of this peptide to interfere in MII-Gt interactions is paradoxically low compared with its activity to stabilize MII. Besides this disparity, the pH profiles of competition with Gt are characteristically different for the two peptides. We propose a two-site sequential fit model for signal transfer from the activated receptor, R*, to the G-protein. In the center of the model is specific recognition of conformationally distinct sites of R* by Gtalpha(340-350) and Gtgamma(50-71)farnesyl. One matching pair of domains on the proteins would, on binding, lead to a conformational change in the G-protein and/or receptor, with subsequent binding of the second pair of domains. This process could be the structural basis for GDP release and the formation of a stable empty site complex that is ready to receive the activating cofactor, GTP.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Células Fotorreceptoras/metabolismo , Rodopsina/metabolismo , Animais , Sítios de Ligação , Proteínas de Ligação ao GTP/química , Modelos Moleculares , Ligação Proteica , Rodopsina/química , Transdução de SinaisRESUMO
Purified bovine rhodopsin solubilized in dodecyl maltoside was photolyzed at 20 degreesC with 477 nm light, and difference spectra were collected at time delays ranging from 10 micros to 10 ms after photolysis. Bromocresol purple was added to the samples to detect pH changes in the aqueous environment due to changes in the protonation state of rhodopsin. The data were analyzed using singular value decomposition and global exponential fitting, which revealed three exponential processes indicating the presence of at least four intermediates. Spectral changes of the indicator dye were separated from those of rhodopsin, and proton release and uptake rates were analyzed within the framework of rhodopsin photoreaction kinetics. Proton release occurred during Lumi decay to Meta-I380 followed by uptake upon Meta-I380 decay and by a more significant proton uptake with the time course of Meta-I480 decay. On the basis of the estimated number of protons released and taken up in each step of the rhodopsin photoreaction, we concluded that two forms of Meta-II are present. The two forms of Meta-II, Meta-IIa' and Meta-IIb, differ in protonation state from one another as do both from the earlier, 380 nm absorbing form, Meta-I380.
Assuntos
Prótons , Rodopsina/metabolismo , Absorção , Animais , Púrpura de Bromocresol/metabolismo , Bovinos , Corantes/metabolismo , Transferência de Energia , Indicadores e Reagentes/metabolismo , Cinética , Fotólise , Rodopsina/química , EspectrofotometriaRESUMO
Fast stochastic equilibrium fluctuations (time scale: 10(-10)-10(-13) seconds) in purple membranes (MP) and in disk membranes (DM) have been measured with quasielastic incoherent neutron scattering. The comparison of predominantly stochastic motions occurring in purple membranes and in disk membranes revealed qualitatively similar dynamical behaviour. Models of internal motions within restricted volumes have been shown to be useful to fit the spectra from both samples. From fits using these models we found "amplitudes" 15 to 20% larger for motions in DM samples compared to PM samples. This indicates a higher internal flexibility of the DM. Because the dynamical behaviour is very sensitive to the hydration of the protein-lipid complex, we also performed neutron diffraction experiments to determine lamellar spacings as a measure of level of hydration and as a function of temperature. From these studies the interaction of solvent molecules with the surface of the protein-lipid complex appears to be qualitatively similar for both types of membranes.
Assuntos
Membranas Artificiais , Membrana Purpúrea/química , Água/química , Algoritmos , Difusão , Transferência de Energia , Halobacterium/química , Nêutrons , Espalhamento de Radiação , TemperaturaRESUMO
Rhodopsin-transducin coupling was used as an assay to investigate a laterally patterned membrane reconstituted with a receptor and its G protein. It served as a model system to show the feasibility to immobilize G protein-coupled receptors on solid supports and investigate receptor activation and interaction with G proteins by one-dimensional imaging surface plasmon resonance. Supported membranes were formed by the self-assembly of lipids and rhodopsin from detergent solution onto functionalized gold surfaces. They formed micrometer-sized alternating regions of pure fluid phospholipid bilayers separated by bilayers composed of an outer phospholipid leaflet on a gold-attached inner thiolipid. Rhodopsin was found to incorporate preferentially into the phospholipid bilayer regions, whereas transducin was uniformly distributed over the entire outer surface of the supported patterned membrane. The influence of rhodopsin on the dark binding of transducin to lipid membranes was described quantitatively and compared with previously published data. Coupling reactions with transducin resembled closely the native system, indicating that the native functionality of rhodopsin was preserved in the supported membranes. The spatially varying properties of the membranes resulted in a pattern of rhodopsin activity on the surface. This combination of techniques is very promising for the investigation of the lateral diffusion of transducin, can be extended to include signalling proteins downstream of the G protein, and may be applied to functional screening of other G protein-coupled receptors. In the future, it may also serve as a basis for constructing biosensors based on receptor proteins.
Assuntos
Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Rodopsina/metabolismo , Transducina/metabolismo , Técnicas Biossensoriais , Fluidez de Membrana , Fosfolipídeos , Ligação Proteica/efeitos da radiação , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Compostos de SulfidrilaRESUMO
The photoreceptor rhodopsin is a seven-transmembrane helix receptor that activates the G protein transducin in response to light. Several site-directed rhodopsin mutants have been reported to be defective in transducin activation. Two of these mutants bound transducin in response to light, but failed to release the bound transducin in the presence of GTP (Franke, R. R., König, B., Sakmar, T. P., Khorana, H. G., and Hofmann, K. P. (1990) Science 250, 123-125). The present study was carried out to determine the nucleotide-binding state of transducin as it interacts with rhodopsin mutants. Five mutant bovine opsin genes were prepared by site-specific mutagenesis. Three mutant genes had deletions from one cytoplasmic loop each: AB delta 70-71; CD delta 143-150; and EF delta 237-249. Two additional loop CD mutant genes were prepared: E134R/R135E had a reversal of a conserved charge pair, and CD r140-152 had a 13-amino acid sequence replaced by a sequence derived from the amino-terminal tail. Three types of assays were carried out: 1) a fluorescence assay of photoactivated rhodopsin (R*)-dependent guanosine 5'-O-(3-thiotriphosphate) uptake by transducin, 2) an assay of R*-dependent release of labeled GDP from the alpha-subunit of transducin holoenzyme (Gt alpha).GDP, and 3) a light-scattering assay of R*.Gt complex formation and dissociation. We show that the mutant pigments, which are able to bind transducin in a light-dependent manner but lack the ability to activate transducin, most likely form R*.Gt alpha beta gamma.GDP complexes that are impaired in GDP release.