RESUMEN
Class I benzoyl-CoA reductases (BCRs) are oxygen-sensitive key enzymes in the degradation of monocyclic aromatic compounds in anaerobic prokaryotes. They catalyze the ATP-dependent reductive dearomatization of their substrate to cyclohexa-1,5-diene-1-carboxyl-CoA (1,5-dienoyl-CoA). An aromatizing 1,5-dienoyl-CoA oxidase (DCO) activity has been proposed to protect BCRs from oxidative damage, however, the gene and its product involved have not been identified, yet. Here, we heterologously produced a DCO from the hyperthermophilic euryarchaeon Ferroglobus placidus that coupled the oxidation of two 1,5-dienoyl-CoA to benzoyl-CoA to the reduction of O2 to water at 80°C. DCO showed similarities to members of the old yellow enzyme family and contained FMN, FAD and an FeS cluster as cofactors. The O2 -dependent activation of inactive, reduced DCO is assigned to a redox thiol switch at Eo ' = -3 mV. We propose a catalytic cycle in which the active site FMN/disulfide redox centers are reduced by two 1,5-dienoyl-CoA (reductive half-cycle), followed by two consecutive two-electron transfer steps to molecular oxygen via peroxy- and hydroxyflavin intermediates yielding water (oxidative half-cycle). This work identified the enzyme involved in a unique oxygen detoxification process for an oxygen-sensitive catabolic enzyme.
Asunto(s)
Archaeoglobales/metabolismo , Metabolismo Energético/fisiología , Hidroliasas/metabolismo , Hidrocarburos Aromáticos/metabolismo , Oxígeno/metabolismo , Archaeoglobales/enzimología , Archaeoglobales/genética , Dominio Catalítico/fisiología , Disulfuros/metabolismo , Flavinas/metabolismo , Hidroliasas/genética , Hidrólisis , Oxidación-ReducciónRESUMEN
In the degenerative eye disease retinitis pigmentosa (RP), protein misfolding leads to fatal consequences for cell metabolism and rod and cone cell survival. To stop disease progression, a therapeutic approach focuses on stabilizing inherited protein mutants of the G protein-coupled receptor (GPCR) rhodopsin using pharmacological chaperones (PC) that improve receptor folding and trafficking. In this study, we discovered stabilizing nonretinal small molecules by virtual and thermofluor screening and determined the crystal structure of pharmacologically stabilized opsin at 2.4 Å resolution using one of the stabilizing hits (S-RS1). Chemical modification of S-RS1 and further structural analysis revealed the core binding motif of this class of rhodopsin stabilizers bound at the orthosteric binding site. Furthermore, previously unobserved conformational changes are visible at the intradiscal side of the seven-transmembrane helix bundle. A hallmark of this conformation is an open channel connecting the ligand binding site with the membrane and the intradiscal lumen of rod outer segments. Sufficient in size, the passage permits the exchange of hydrophobic ligands such as retinal. The results broaden our understanding of rhodopsin's conformational flexibility and enable therapeutic drug intervention against rhodopsin-related retinitis pigmentosa.
Asunto(s)
Diseño de Fármacos , Preparaciones Farmacéuticas/administración & dosificación , Conformación Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Receptores Acoplados a Proteínas G/química , Rodopsina/química , Animales , Células Cultivadas , Humanos , Ligandos , Ratones , Modelos Moleculares , Preparaciones Farmacéuticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Rodopsina/metabolismoRESUMEN
A reaction microscope dedicated to multi-particle coincidence spectroscopy on gas-phase samples is installed at beamline FL26 of the free-electron laser FLASH2 in Hamburg. The main goals of the instrument are to follow the dynamics of atoms, molecules and small clusters on their natural time-scale and to study non-linear light-matter interaction with such systems. To this end, the reaction microscope is combined with an in-line extreme-ultraviolet (XUV) split-delay and focusing optics, which allows time-resolved XUV-XUV pump-probe spectroscopy to be performed.
RESUMEN
Time delays for atomic photoemission obtained in streaking or reconstruction of attosecond bursts by interference of two-photon transitions experiments originate from a combination of the quantum mechanical Wigner time and the Coulomb-laser coupling. While the former was investigated intensively theoretically as well as experimentally, the latter attracted less interest in experiments and has mostly been subject to calculations. Here, we present a measurement of the Coulomb-laser coupling-induced time shifts in photoionization of neon at 59.4 eV using a terahertz (THz) streaking field (λ=152 µm). Employing a reaction microscope at the THz beamline of the free-electron laser in Hamburg (FLASH), we have measured relative time shifts of up to 70 fs between the emission of 2p photoelectrons (â¼38 eV) and low-energetic (<1 eV) photoelectrons. A comparison with theoretical predictions on Coulomb-laser coupling reveals reasonably good agreement.
RESUMEN
Charge transfer (CT) at avoided crossings of excited ionized states of argon dimers is observed using a two-color pump-probe experiment at the free-electron laser in Hamburg (FLASH). The process is initiated by the absorption of three 27-eV-photons from the pump pulse, which leads to the population of Ar2+*-Ar states. Due to nonadiabatic coupling between these one-site doubly ionized states and two-site doubly ionized states of the type Ar+*-Ar+, CT can take place leading to the population of the latter states. The onset of this process is probed by a delayed infrared (800 nm) laser pulse. The latter ionizes the dimers populating repulsive Ar2+ -Ar+ states, which then undergo a Coulomb explosion. From the delay-dependent yields of the obtained Ar2+ and Ar+ ions, the lifetime of the charge-transfer process is extracted. The obtained experimental value of (531 ± 136) fs agrees well with the theoretical value computed from Landau-Zener probabilities.
RESUMEN
The class I benzoyl-coenzyme A (BzCoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds that catalyze the ATP-dependent dearomatization of their substrate to a cyclic dienoyl-CoA. The phylogenetically distinct Thauera- and Azoarcus-type BCR subclasses are iron-sulfur enzymes and consist of an ATP-hydrolyzing electron activation module and a BzCoA reduction module. More than 20 years after their initial identification, all biochemical information about class I BCRs derives from studies of the wild-type enzyme from the denitrifying bacterium Thauera aromatica (BCRTaro). Here, we describe the first heterologous production and purification of the ATP-hydrolyzing, electron-activating module of an Azoarcus-type BCR from the hyperthermophilic archaeon Ferroglobus placidus, BzdPQFpla. The Fe content, UV/vis spectroscopic, and Mössbauer spectroscopic properties of the 57Fe-enriched enzyme clearly identified a [4Fe-4S]+/2+ cluster with a redox potential (E°') of -376 mV as a cofactor. ATP hydrolysis is required to overcome a redox barrier of â¼250 mV for stoichiometric electron transfer from the [4Fe-4S]+ cluster to the substrate benzene ring (E°'BzCoA/dienoyl-CoA = -622 mV). BzdPQFpla exhibited ATPase activity (15 nmol min-1 mg-1; Km = 270 µM) at 75 °C, which was relatively stable in air in contrast to BCRTaro. The results obtained revealed high levels of functional and molecular similarity between Azoarcus-type BCRs and the homologous ATP-dependent activator components of 2-hydroxyacyl-CoA dehydratases involved in amino acid fermentations. Insights into the diversity and evolution of ATP-dependent electron-activating modules for catalytic or stoichiometric low-potential electron transfer processes are presented.
Asunto(s)
Adenosina Trifosfato/metabolismo , Archaea/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Catálisis , Electrones , Escherichia coli/genética , Proteínas Hierro-Azufre/metabolismo , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The fragmentation of free tenfold protonated ubiquitin in intense 70â femtosecond pulses of 90â eV photons from the FLASH facility was investigated. Mass spectrometric investigation of the fragment cations produced after removal of many electrons revealed fragmentation predominantly into immonium ions and related ions, with yields increasing linearly with intensity. Ionization clearly triggers a localized molecular response that occurs before the excitation energy equilibrates. Consistent with this interpretation, the effect is almost unaffected by the charge state, as fragmentation of sixfold deprotonated ubiquitin leads to a very similar fragmentation pattern. Ubiquitin responds to EUV multiphoton ionization as an ensemble of small peptides.
RESUMEN
The Fe(III)-respiring Ferroglobus placidus is the only known archaeon and hyperthermophile for which a complete degradation of aromatic substrates to CO2 has been reported. Recent genome and transcriptome analyses proposed a benzoyl-coenzyme A (CoA) degradation pathway similar to that found in the phototrophic Rhodopseudomonas palustris, which involves a cyclohex-1-ene-1-carboxyl-CoA (1-enoyl-CoA) forming, ATP-dependent key enzyme benzoyl-CoA reductase (BCR). In this work, we demonstrate, by first in vitro studies, that benzoyl-CoA is ATP-dependently reduced by two electrons to cyclohexa-1,5-dienoyl-CoA (1,5-dienoyl-CoA), which is further degraded by hydration to 6-hydroxycyclohex-1-ene-1-carboxyl-CoA (6-OH-1-enoyl-CoA); upon addition of NAD(+) , the latter was subsequently converted to ß-oxidation intermediates. The four candidate genes of BCR were heterologously expressed, and the enriched, oxygen-sensitive enzyme catalysed the two-electron reduction of benzoyl-CoA to 1,5-dienoyl-CoA. A gene previously assigned to a 2,3-didehydropimeloyl-CoA hydratase was heterologously expressed and shown to act as a typical 1,5-dienoyl-CoA hydratase that does not accept 1-enoyl-CoA. A gene previously assigned to a 1-enoyl-CoA hydratase was heterologously expressed and identified to code for a bifunctional crotonase/3-OH-butyryl-CoA dehydrogenase. In summary, the results consistently provide biochemical evidence that F. placidus and probably other archaea predominantly degrade aromatics via the Thauera/Azoarcus type and not or only to a minor extent via the predicted R. palustris-type benzoyl-CoA degradation pathway.
Asunto(s)
Acilcoenzima A/metabolismo , Archaeoglobales/enzimología , Redes y Vías Metabólicas/fisiología , Anaerobiosis , Archaeoglobales/genética , Coenzima A/metabolismo , Enoil-CoA Hidratasa/metabolismo , Compuestos Férricos/metabolismo , Hidroliasas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Thauera/metabolismoRESUMEN
The human ether-a-go-go related gene (hERG) potassium channel plays a major role in the repolarization of the cardiac action potential. Inhibition of the hERG function by mutations or a wide variety of pharmaceutical compounds cause long QT syndrome and lead to potentially lethal arrhythmias. For detailed insights into the structural and biochemical background of hERG function and drug binding, the purification of recombinant protein is essential. Because the hERG channel is a challenging protein to purify, fast and easy techniques to evaluate different expression, solubilization and purification conditions are of primary importance. Here, we describe the generation of a set of 12 monoclonal antibodies against hERG. Beside their suitability in western blot, immunoprecipitation and immunostaining, these antibodies were used to establish a sandwich ELISA for the detection and relative quantification of hERG in different expression systems. Furthermore, a Fab fragment was used in fluorescence size exclusion chromatography to determine the oligomeric state of hERG after solubilization. These new tools can be used for a fast and efficient screening of expression, solubilization and purification conditions.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Canales de Potasio Éter-A-Go-Go/análisis , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Cromatografía en Gel/métodos , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/inmunología , Canales de Potasio Éter-A-Go-Go/aislamiento & purificación , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas , RatonesRESUMEN
Facing rapid fluctuations in their natural environment, extremophiles, like the hyperthermophilic archaeon Pyrococcus furiosus, exhibit remarkable adaptability to extreme conditions. However, our understanding of their dynamic cellular responses remains limited. This study integrates RNA-sequencing and mass spectrometry data, thereby elucidating transcriptomic and proteomic responses to heat and cold shock stress in P. furiosus. Our results reveal rapid and dynamic changes in gene and protein expression following these stress responses. Heat shock triggers extensive transcriptome reprogramming, orchestrated by the transcriptional regulator Phr, targeting a broader gene repertoire than previously demonstrated. For heat shock signature genes, RNA levels swiftly return to baseline upon recovery, while protein levels remain persistently upregulated, reflecting a rapid but sustained response. Intriguingly, cold shock at 4°C elicits distinct short- and long-term responses at both RNA and protein levels. Cluster analysis identified gene sets with either congruent or contrasting trends in RNA and protein changes, representing well-separated arCOG groups tailored to their individual cellular responses. Particularly, upregulation of ribosomal proteins and significant enrichment of 5'-leadered sequences in cold-shock responsive genes suggest that translation regulation is important during cold shock adaption. Further investigating transcriptomic features, we reveal that thermal stress genes are equipped with basal sequence elements, such as strong promoter and poly(U)-terminators, facilitating a regulated response of the respective transcription units. Our study provides a comprehensive overview of the cellular response to temperature stress, advancing our understanding of stress response mechanisms in hyperthermophilic archaea and providing valuable insights into the molecular adaptations that facilitate life in extreme environments.IMPORTANCEExtreme environments provide unique challenges for life, and the study of extremophiles can shed light on the mechanisms of adaptation to such conditions. Pyrococcus furiosus, a hyperthermophilic archaeon, is a model organism for studying thermal stress response mechanisms. In this study, we used an integrated analysis of RNA-sequencing and mass spectrometry data to investigate the transcriptomic and proteomic responses of P. furiosus to heat and cold shock stress and recovery. Our results reveal the rapid and dynamic changes in gene and protein expression patterns associated with these stress responses, as well as the coordinated regulation of different gene sets in response to different stressors. These findings provide valuable insights into the molecular adaptations that facilitate life in extreme environments and advance our understanding of stress response mechanisms in hyperthermophilic archaea.
RESUMEN
Upon ionization, water forms a highly acidic radical cation H2O+· that undergoes ultrafast proton transfer (PT)-a pivotal step in water radiation chemistry, initiating the production of reactive H3O+, OH[Formula: see text] radicals, and a (hydrated) electron. Until recently, the time scales, mechanisms, and state-dependent reactivity of ultrafast PT could not be directly traced. Here, we investigate PT in water dimers using time-resolved ion coincidence spectroscopy applying a free-electron laser. An extreme ultraviolet (XUV) pump photon initiates PT, and only dimers that have undergone PT at the instance of the ionizing XUV probe photon result in distinct H3O+ + OH+ pairs. By tracking the delay-dependent yield and kinetic energy release of these ion pairs, we measure a PT time of (55 ± 20) femtoseconds and image the geometrical rearrangement of the dimer cations during and after PT. Our direct measurement shows good agreement with nonadiabatic dynamics simulations for the initial PT and allows us to benchmark nonadiabatic theory.
RESUMEN
The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-ß-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-ß-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [³H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [³H]neutral sterols and [³H]bile acids, whereas all compounds increased plasma HDL-[³H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-ß-HDL formation, which may be required to increase reverse cholesterol transport.
Asunto(s)
Anticolesterolemiantes/farmacología , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Colesterol/metabolismo , Lipoproteínas de Alta Densidad Pre-beta/metabolismo , Amidas , Animales , Ácidos y Sales Biliares/metabolismo , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Colesterol/sangre , Proteínas de Transferencia de Ésteres de Colesterol/sangre , Cricetinae , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Ésteres , Lipoproteínas de Alta Densidad Pre-beta/sangre , Humanos , Oxazolidinonas/farmacología , Quinolinas/farmacología , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Pyrococcus furiosus is a model organism for analyses of molecular biology and biochemistry of archaea, but so far no useful genetic tools for this species have been described. We report here a genetic transformation system for P. furiosus based on the shuttle vector system pYS2 from Pyrococcus abyssi. In the redesigned vector, the pyrE gene from Sulfolobus was replaced as a selectable marker by the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (HMG-CoA) conferring resistance of transformants to the antibiotic simvastatin. Use of this modified plasmid resulted in the overexpression of the HMG-CoA reductase in P. furiosus, allowing the selection of strains by growth in the presence of simvastatin. The modified shuttle vector replicated in P. furiosus, but the copy number was only one to two per chromosome. This system was used for overexpression of His(6)-tagged subunit D of the RNA polymerase (RNAP) in Pyrococcus cells. Functional RNAP was purified from transformed cells in two steps by Ni-NTA and gel filtration chromatography. Our data provide evidence that expression of transformed genes can be controlled from a regulated gluconeogenetic promoter.
Asunto(s)
Vectores Genéticos/genética , Pyrococcus furiosus/genética , Transformación Genética , Acilcoenzima A , Cromatografía de Afinidad , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/metabolismo , Dosificación de Gen , Regulación Bacteriana de la Expresión Génica , Plásmidos/genéticaRESUMEN
Protein-protein interactions (PPIs) are at the core of virtually all biological processes in cells. Consequently, targeting PPIs is emerging at the forefront of drug discovery. Cellular assays which closely recapitulate native conditions in vivo are instrumental to understand how small molecule drugs can modulate such interactions. We have integrated MultiBacMam, a baculovirus-based mammalian gene delivery tool we developed, with bimolecular fluorescence complementation (BiFC), giving rise to a highly efficient system for assay development, identification and characterization of PPI modulators. We used our system to analyze compounds impacting on CDK5-p25 PPI, which is implicated in numerous diseases including Alzheimer's. We evaluated our tool-kit with the known inhibitor p5T, and we established a mini-screen to identify compounds that modulate this PPI in dose-response experiments. Finally, we discovered several compounds disrupting CDK5-p25 PPI, which had not been identified by other screening or structure-based methods before.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Proteínas del Tejido Nervioso/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Descubrimiento de Drogas/métodos , Fluorescencia , Humanos , Simulación del Acoplamiento Molecular , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
OBJECTIVE: Schizophrenia is associated with increased cardiovascular mortality. The deceleration capacity of heart rate is discussed to be a predictor of mortality, more powerful than conventional measures of heart rate variability (HRV) or the left ventricular ejection fraction. The aim of this study was to determine whether patients with schizophrenia, receiving antipsychotic medication have a reduced HRV indicating an elevated mortality risk. METHODS: We quantified HRV and the deceleration capacity in 24-hour electrocardiogram recordings from 28 medicated patients with schizophrenia and 28 matched controls. In addition to the evaluation of the 24-hour recording, 4-hour periods of "sleep" and "wake" recordings were evaluated separately, as activity has a major influence on HRV. Actigraphy was used to identify coherent sleep and wake phases and to ensure comparable levels of activity in patients and controls. RESULTS: Medicated patients showed a significant reduction of the HRV. The HRV was reduced significantly according to the time domain and frequency domain measures. The deceleration capacity of medicated patients was 5.36 compared with 8.26 for the controls (p < .05). CONCLUSIONS: Heart rate deceleration capacity is significantly reduced in schizophrenic patients treated with antipsychotics and may serve as an indicator of increased cardiovascular mortality risk.
Asunto(s)
Antipsicóticos/efectos adversos , Enfermedades Cardiovasculares/inducido químicamente , Enfermedades Cardiovasculares/mortalidad , Frecuencia Cardíaca/efectos de los fármacos , Síndrome de QT Prolongado/inducido químicamente , Síndrome de QT Prolongado/mortalidad , Esquizofrenia Hebefrénica/tratamiento farmacológico , Esquizofrenia Hebefrénica/mortalidad , Esquizofrenia Paranoide/tratamiento farmacológico , Esquizofrenia Paranoide/mortalidad , Adulto , Antipsicóticos/uso terapéutico , Causas de Muerte , Muerte Súbita Cardíaca/epidemiología , Muerte Súbita Cardíaca/etiología , Quimioterapia Combinada , Electrocardiografía Ambulatoria/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Actividad Motora/efectos de los fármacos , Factores de Riesgo , Procesamiento de Señales Asistido por ComputadorRESUMEN
Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed ß-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines.
Asunto(s)
Anticuerpos Antiprotozoarios/química , Anticuerpos Antiprotozoarios/metabolismo , Antígenos de Protozoos/química , Antígenos de Protozoos/metabolismo , Vacunas contra la Malaria/química , Vacunas contra la Malaria/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
In mass spectroscopic experiments of oxygen evolution in Photosystem II at 50% enrichment of H(2)18O, one expects equal signals of 18O(2) and 16O(2) unless one of the isotopes is favored by the oxygen evolving complex (OEC). We have observed a deviation from this expectation, being a clear indication of an isotope effect. We have measured the effect to be 1.14-1.30, which is higher than the theoretically predicted value of 1.014-1.06. This together with the strong temperature variation of the measured effect with a discontinuity at 11 degrees C observed for wild-type tobacco and at 9 degrees C for a yellow-green tobacco mutant suggest that an additional mechanism is responsible for the observed high isotope effect. The entry of a finite size of water clusters to the cleavage site of the OEC can explain the observation.
Asunto(s)
Nicotiana/metabolismo , Isótopos de Oxígeno , Oxígeno/química , Fotosíntesis , Agua/química , Espectrometría de Masas , Oxígeno/metabolismo , Temperatura , Tilacoides/química , Factores de Tiempo , Nicotiana/química , Agua/metabolismoRESUMEN
After 25 years of intensive research, the understanding of how photoreceptors in the eye perceive light and convert it into nerve signals has largely advanced. Central to this is the structural and mechanistic exploration of the G protein-coupled receptor rhodopsin acting as a dim-light sensing pigment in the retina. Investigation of rhodopsin by X-ray crystallographic, electron microscopic, and biochemical means depends on the ability to produce and isolate pure rhodopsin protein. Robust and well-defined protocols permit the production and crystallization of rhodopsin variants to investigate the inactive ground, the fully activated metarhodopsin II state, or disease-causing rhodopsin mutations. This chapter details how we express and purify biologically active variants of rhodopsin from HEK293S GnTI(-) cells in a quality and quantity suitable for biochemical assays, crystallization, and structure determination.
Asunto(s)
Rodopsina/química , Línea Celular , Cristalografía por Rayos X , Humanos , Microscopía Electrónica , Retina/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Rodopsina/ultraestructuraRESUMEN
Spin and valence states of the non-heme iron and the heme iron of cytochrome b559, as well as their interactions with alpha-tocopherol quinone (alpha-TQ) in photosystem II (PSII) thylakoid membranes prepared from the Chlamydomonas reinhardtii PSI- mutant have been studied using Mössbauer spectroscopy. Both of the iron atoms are in low spin ferrous states. The Debye temperature of the non-heme is 194 K and of the heme iron is 182 K. The treatment of alpha-TQ does not change the spin and the valence states of the non-heme iron but enhances the covalence of its bonds. alpha-TQ oxidizes the heme iron into the high spin Fe3+ state. A possible role of the non-heme iron and alpha-TQ in electron flow through the PSII is discussed.
Asunto(s)
Chlamydomonas reinhardtii/química , Grupo Citocromo b/química , Hierro/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Complejo de Proteína del Fotosistema II , Vitamina E/análogos & derivados , Vitamina E/química , Animales , Chlamydomonas reinhardtii/genética , Hemo/química , Immunoblotting , Isótopos de Hierro , Modelos Químicos , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Espectroscopía de Mossbauer , Temperatura , Tilacoides/químicaRESUMEN
We have found that copper(II) ions at about equimolar Cu2+/photosystem II (PS II) reaction center proportions stimulate oxygen evolution nearly twofold. This high affinity Cu-binding site is different from the binding sites of Mn and Ca ions. The analysis of the Cu2+ content in PS II preparations isolated from wild-type tobacco and a tobacco mutant deficient in light-harvesting complex suggests that Cu2+ may be a native component of PS II and may take part in the oxygen evolution process. At higher concentrations, Cu2+ ions inhibit oxygen evolution and quench fluorescence.