RESUMEN
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.
Asunto(s)
Rodopsina , Visión Ocular , Animales , Sitios de Unión/efectos de la radiación , Cristalografía , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Isomerismo , Fotones , Unión Proteica/efectos de la radiación , Conformación Proteica/efectos de la radiación , Retinaldehído/química , Retinaldehído/metabolismo , Retinaldehído/efectos de la radiación , Rodopsina/química , Rodopsina/metabolismo , Rodopsina/efectos de la radiación , Factores de Tiempo , Visión Ocular/fisiología , Visión Ocular/efectos de la radiaciónRESUMEN
Animal vision depends on opsins, a category of G protein-coupled receptor (GPCR) that achieves light sensitivity by covalent attachment to retinal. Typically binding as an inverse agonist, 11-cis retinal photoisomerizes to the all-trans isomer and activates the receptor, initiating downstream signaling cascades. Retinal bound to bistable opsins isomerizes back to the 11-cis state after absorption of a second photon, inactivating the receptor. Bistable opsins are essential for invertebrate vision and nonvisual light perception across the animal kingdom. While crystal structures are available for bistable opsins in the inactive state, it has proven difficult to form homogeneous populations of activated bistable opsins either via illumination or reconstitution with all-trans retinal. Here, we show that a nonnatural retinal analog, all-trans retinal 6.11 (ATR6.11), can be reconstituted with the invertebrate bistable opsin, Jumping Spider Rhodopsin-1 (JSR1). Biochemical activity assays demonstrate that ATR6.11 functions as a JSR1 agonist. ATR6.11 binding also enables complex formation between JSR1 and signaling partners. Our findings demonstrate the utility of retinal analogs for biophysical characterization of bistable opsins, which will deepen our understanding of light perception in animals.
Asunto(s)
Opsinas , Retinaldehído , Animales , Retinaldehído/metabolismo , Retinaldehído/química , Retinaldehído/análogos & derivados , Opsinas/metabolismo , Opsinas/química , Rodopsina/metabolismo , Rodopsina/química , Arañas/metabolismo , HumanosRESUMEN
G protein-coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR-G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand-GPCR-partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [ß1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using ß1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gαißγ and ß-arrestin-1 and showed that carvedilol induces an increase in coupling of ß-arrestin-1 and Gαißγ to ß1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores Opioides/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Arrestina/genética , Arrestina/metabolismo , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ligandos , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores Opioides/química , Anticuerpos de Cadena Única , Pavos , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography, the details of ligand-induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey ß1-adrenergic receptor (ß1AR). Labelling with [(15)N]valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental method to delineate signal transmission networks at high resolution in GPCRs.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/metabolismo , Transducción de Señal , Agonistas de Receptores Adrenérgicos beta 1/química , Agonistas de Receptores Adrenérgicos beta 1/farmacología , Antagonistas de Receptores Adrenérgicos beta 1/farmacología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Animales , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Agonismo Parcial de Drogas , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Ligandos , Modelos Moleculares , Movimiento , Mutación Puntual/genética , Estabilidad Proteica , Estructura Secundaria de Proteína/efectos de los fármacos , Receptores Adrenérgicos beta 1/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , PavosRESUMEN
In this work, we examine methyl nuclear magnetic resonance (NMR) spectra of the methionine ε-[13CH3] labelled thermostabilized ß1 adrenergic receptor from turkey in association with a variety of different effectors, including mini-Gs and nanobody 60 (Nb60), which have not been previously studied in complex with ß1 adrenergic receptor (ß1AR) by NMR. Complexes with pindolol and Nb60 induce highly similar inactive states of the receptor, closely resembling the resting state conformational ensemble. We show that, upon binding of mini-Gs or nanobody 80 (Nb80), large allosteric changes throughout the receptor take place. The conformation of tß1AR stabilized by the native-like mini-Gs protein is highly similar to the conformation induced by the currently used surrogate Nb80. Interestingly, in both cases residual dynamics are present, which were not observed in the resting states. Finally, we reproduce a pharmaceutically relevant situation, where an antagonist abolishes the interaction of the receptor with the mini-G protein in a competitive manner, validating the functional integrity of our preparation. The presented system is therefore well suited for reproducing the individual steps of the activation cycle of a G protein-coupled receptor (GPCR) in vitro and serves as a basis for functional and pharmacological characterizations of more native-like systems in the future.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Pindolol/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Anticuerpos de Cadena Única/metabolismo , Anticuerpos de Dominio Único/inmunología , Sitios de Unión , Cristalografía por Rayos X , Humanos , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , TurquíaRESUMEN
The binding of imidazolium salts to cucurbit[8]uril, CB[8], triggers a stepwise self-assembly process with semiflexible polymer chains and crystalline nanostructures as early- and late-stage species, respectively. In such a process, which involves the crystallization of the host-guest complexes, the guest plays a critical role in directing self-assembly toward desirable morphologies. These include platelet-like aggregates and two-dimensional (2D) fibers, which, moreover, exhibit viscoelastic and lyotropic properties. Our observations provide a deeper understanding of the self-assembly of CB[8] complexes, with fundamental implications in the design of functional 2D systems and crystalline materials.
RESUMEN
Resistance to the current therapies is the main clinical challenge in the treatment of lethal metastatic prostate cancer (mPCa). Developing novel therapeutic approaches with effective regimes and minimal side effects for this fatal disease remain a priority in prostate cancer study. In the present study, we demonstrated that a traditional Chinese medicine, quality-assured Ganoderma tsugae ethanol extract (GTEE), significantly suppressed cell growth and metastatic capability and caused cell cycle arrest through decreasing expression of cyclins in mPCa cells, PC-3 and DU145 cells. GTEE also induced caspase-dependent apoptosis in mPCa cells. We further showed the potent therapeutic efficacy of GTEE by inhibiting subcutaneous PC-3 tumor growth in a xenograft model. The in vitro and in vivo efficacies on mPCa cells were due to blockade of the PI3K/Akt and MAPK/ERK signaling pathways associated with cancer cell growth, survival and apoptosis. These preclinical data provide the molecular basis for a new potential therapeutic approach toward the treatment of lethal prostate cancer progression.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Medicamentos Herbarios Chinos/farmacología , Ganoderma/química , Animales , Antineoplásicos Fitogénicos/química , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Medicina Tradicional China , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.
RESUMEN
The numerous chemokines and their cognate G protein-coupled chemokine receptors on the surface of leukocytes form a complex signaling network, which regulates the immune response and also other key physiological processes. Currently only a very limited number of structures of chemokineâ¢chemokine receptor complexes have been solved. More structures are needed for the understanding of their mechanism of action and the rational design of drugs against these highly relevant therapeutic targets. Recently, we have determined the cryo-EM structure of the human wild-type CCR5 chemokine receptor, which is also the HIV-1 coreceptor, in its active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist CCR5 chemokine ligands. In this chapter, we present a detailed protocol for the preparation of the active agonist chemokineâ¢CCR5â¢Gi complex for cryo-EM studies including quality controls and caveats. As such the protocol may serve as starting point for structural and biophysical studies of other chemokineâ¢chemokine receptor complexes.
Asunto(s)
Receptores CCR5 , Transducción de Señal , Quimiocina CCL5/química , Quimiocinas/metabolismo , Microscopía por Crioelectrón , Humanos , Receptores CCR5/química , Receptores CCR5/metabolismo , Receptores Acoplados a Proteínas GRESUMEN
The complex of G protein-coupled receptors (GPCR) and G proteins is the core assembly in GPCR signaling in eukaryotes. With the recent development of cryo-electron microscopy, there has been a rapid growth in structures of GPCR-G protein complexes solved to near-atomic resolution, giving important insights into this signaling complex. Here we describe the biochemical protocol to study the interaction between GPCRs and G proteins before preparation of GPCR-G protein complexes for structural studies. We use gel filtration to analyze the binding properties between GPCR and G protein with the presence of agonist or antagonist, as well as the complex dissociation in the presence of GTP analogue. Methods used in the protocol are affinity purification and gel filtration, which are also commonly used in protein sample preparation for structural work. Therefore, the protocol can be easily adapted for large-scale sample preparation.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Guanosina Trifosfato/análogos & derivados , Receptores Acoplados a Proteínas G/metabolismo , Membrana Celular/metabolismo , Cromatografía en Gel , Proteínas de Unión al GTP/química , Células HEK293 , Humanos , Unión Proteica , Receptores Acoplados a Proteínas G/química , Transducción de SeñalRESUMEN
Serial data collection has emerged as a major tool for data collection at state-of-the-art light sources, such as microfocus beamlines at synchrotrons and X-ray free-electron lasers. Challenging targets, characterized by small crystal sizes, weak diffraction and stringent dose limits, benefit most from these methods. Here, the use of a thin support made of a polymer-based membrane for performing serial data collection or screening experiments is demonstrated. It is shown that these supports are suitable for a wide range of protein crystals suspended in liquids. The supports have also proved to be applicable to challenging cases such as membrane proteins growing in the sponge phase. The sample-deposition method is simple and robust, as well as flexible and adaptable to a variety of cases. It results in an optimally thin specimen providing low background while maintaining minute amounts of mother liquor around the crystals. The 2 × 2â mm area enables the deposition of up to several microlitres of liquid. Imaging and visualization of the crystals are straightforward on the highly transparent membrane. Thanks to their affordable fabrication, these supports have the potential to become an attractive option for serial experiments at synchrotrons and free-electron lasers.
Asunto(s)
Cristalografía por Rayos X/métodos , Sustancias Macromoleculares/química , Proteínas/química , Recolección de DatosRESUMEN
The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism.
Asunto(s)
Receptores CCR5/química , Receptores CCR5/metabolismo , Quimiocina CCL5/química , Quimiocina CCL5/metabolismo , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Receptores CCR5/agonistas , Receptores CCR5/genética , Transducción de Señal , Relación Estructura-ActividadRESUMEN
BACKGROUND: Supraglottic jet ventilation (JV(S)) with injectors above airway stenoses may result in inadvertent high lung pressures. We designed this study to investigate intrinsic positive end-expiratory pressure (PEEP(i)) during jet ventilation via a distant injector in a model of dynamic upper airway obstruction. METHODS: Respiratory pressure-time curves were recorded during JV(S) in a tracheal lung model using a pig's trachea and an embolectomy catheter's air-filled balloon to simulate 60 and 80% airway obstruction. JV(S) was performed at various jet frequencies (F(jet) 30 min(-1), 60 min(-1), and 100 min(-1)) and driving pressures (1 bar and 2 bar). RESULTS: JV(S) was associated with generation of PEEP(i), which depended on driving pressure, the degree of obstruction, and on ventilatory frequency. CONCLUSIONS: In the presence of a dynamic upper airway obstruction, JV(S) via a distant injector may result in PEEP(i), which cannot be detected when airway pressure is measured in front of the obstruction.
Asunto(s)
Obstrucción de las Vías Aéreas/terapia , Ventilación con Chorro de Alta Frecuencia , Respiración con Presión Positiva , Animales , Embolectomía , Modelos Anatómicos , Porcinos , Enfermedades de la Tráquea/terapiaRESUMEN
The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 Å resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin-mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin-mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin-mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR-G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.
Asunto(s)
Cristalización/métodos , Proteínas de Unión al GTP/metabolismo , Transducción de SeñalRESUMEN
One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gß subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gß as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.
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Subunidades alfa de la Proteína de Unión al GTP/ultraestructura , Subunidades beta de la Proteína de Unión al GTP/ultraestructura , Subunidades gamma de la Proteína de Unión al GTP/ultraestructura , Rodopsina/ultraestructura , Animales , Bovinos , Microscopía por Crioelectrón , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Complejos Multiproteicos/ultraestructura , Unión Proteica , Rodopsina/metabolismoRESUMEN
Serial femtosecond crystallography of two-dimensional membrane-protein crystals at X-ray free-electron lasers has the potential to address the dynamics of functionally relevant large-scale motions, which can be sterically hindered in three-dimensional crystals and suppressed in cryocooled samples. In previous work, diffraction data limited to a two-dimensional reciprocal-space slice were evaluated and it was demonstrated that the low intensity of the diffraction signal can be overcome by collecting highly redundant data, thus enhancing the achievable resolution. Here, the application of a newly developed method to analyze diffraction data covering three reciprocal-space dimensions, extracting the reciprocal-space map of the structure-factor amplitudes, is presented. Despite the low resolution and completeness of the data set, it is shown by molecular replacement that the reconstructed amplitudes carry meaningful structural information. Therefore, it appears that these intrinsic limitations in resolution and completeness from two-dimensional crystal diffraction may be overcome by collecting highly redundant data along the three reciprocal-space axes, thus allowing the measurement of large-scale dynamics in pump-probe experiments.
RESUMEN
Selective coupling of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) to specific Gα-protein subtypes is critical to transform extracellular signals, carried by natural ligands and clinical drugs, into cellular responses. At the center of this transduction event lies the formation of a signaling complex between the receptor and G protein. We report the crystal structure of light-sensitive GPCR rhodopsin bound to an engineered mini-Go protein. The conformation of the receptor is identical to all previous structures of active rhodopsin, including the complex with arrestin. Thus, rhodopsin seems to adopt predominantly one thermodynamically stable active conformation, effectively acting like a "structural switch," allowing for maximum efficiency in the visual system. Furthermore, our analysis of the well-defined GPCR-G protein interface suggests that the precise position of the carboxyl-terminal "hook-like" element of the G protein (its four last residues) relative to the TM7/helix 8 (H8) joint of the receptor is a significant determinant in selective G protein activation.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Rodopsina/química , Rodopsina/metabolismo , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Mutación , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismo , Rodopsina/genéticaRESUMEN
Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4â Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump-probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.
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The osmoregulated and chill-sensitive glycine-betaine transporter (BetP) from Corynebacterium glutamicum was reconstituted into lipids to form two-dimensional (2D) crystals. The sensitivity of BetP partly bases on its interaction with lipids. Here we demonstrate that lipids and salts influence crystal morphology and crystallinity of a C-terminally truncated BetP. The salt type and concentration during crystallization determined whether crystals grew in the form of planar-tubes, sheets or vesicles, while the lipid type influenced crystal packing and order. Three different lipid preparations for 2D crystallization were compared. Only the use of lipids extracted from C. glutamicum cells led to the formation of large, well-ordered crystalline areas. To understand the lipid-derived influence on crystallinity, lipid extracts from different stages of the crystallization process were analyzed by quantitative multiple-precursor ion scanning mass spectroscopy (MS). Results show that BetP has a preference for fatty acid moieties 16:0-18:1, and that a phosphatidyl glycerol (PG) 16:0-18:1 rich preparation prevents formation of pseudo crystals.