RESUMEN
Ezrin is a member of the ERM (ezrin-radixin-moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ezrin. We observe a bistable array of phenylalanine residues in the core of subdomain F3 that is mobile in the active form and locked in the dormant form. As subdomain F3 is pivotal in binding membrane proteins and phospholipids, these transitions may facilitate activation and signaling. Full-length ezrin forms stable monomers and dimers. We used small-angle X-ray scattering to determine the solution structures of these species. As expected, the monomer shows a globular domain with a protruding helical coiled coil. The dimer shows an elongated dumbbell structure that is twice as long as the monomer. By aligning ERM sequences spanning metazoan evolution, we show that the central helical region is conserved, preserving the heptad repeat. Using this, we have built a dimer model where each monomer forms half of an elongated antiparallel coiled coil with domain-swapped FERM:C-terminal domain complexes at each end. The model suggests that ERM dimers may bind to actin in a parallel fashion.
Asunto(s)
Proteínas del Citoesqueleto/química , Dicroismo Circular , Cristalografía por Rayos X , Dimerización , Conformación ProteicaRESUMEN
The dioxygen we breathe is formed by light-induced oxidation of water in photosystem II. O2 formation takes place at a catalytic manganese cluster within milliseconds after the photosystem II reaction centre is excited by three single-turnover flashes. Here we present combined X-ray emission spectra and diffraction data of 2-flash (2F) and 3-flash (3F) photosystem II samples, and of a transient 3F' state (250 µs after the third flash), collected under functional conditions using an X-ray free electron laser. The spectra show that the initial O-O bond formation, coupled to Mn reduction, does not yet occur within 250 µs after the third flash. Diffraction data of all states studied exhibit an anomalous scattering signal from Mn but show no significant structural changes at the present resolution of 4.5 Å. This study represents the initial frames in a molecular movie of the structural changes during the catalytic reaction in photosystem II.
Asunto(s)
Fotosíntesis/fisiología , Espectrometría por Rayos X/métodos , Agua/metabolismo , Difracción de Rayos X/métodos , Cianobacterias/metabolismo , Modelos Químicos , Oxidación-Reducción , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and without radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract meaningful high-resolution signals from fewer diffraction measurements.
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Rayos Láser , Sustancias Macromoleculares/química , Bacillus/enzimología , Calcio/química , Calibración , Simulación por Computador , Cristalización , Cristalografía por Rayos X , Electrones , Diseño de Equipo , Funciones de Verosimilitud , Modelos Químicos , Conformación Molecular , Muramidasa/química , Nanotecnología , Reproducibilidad de los Resultados , Programas Informáticos , Termolisina/química , Rayos X , Zinc/químicaRESUMEN
In this study we use a combination of absorption, fluorescence and low temperature single-molecule spectroscopy to elucidate the spectral properties, heterogeneities and dynamics of the chlorophyll a (Chla) molecules responsible for the fluorescence emission of photosystem II core complexes (PS II cc) from the cyanobacterium Thermosynechococcus elongatus. At the ensemble level, the absorption and fluorescence spectra show a temperature dependence similar to plant PS II. We report emission spectra of single PS II cc for the first time; the spectra are dominated by zero-phonon lines (ZPLs) in the range between 680 and 705nm. The single-molecule experiments show unambiguously that different emitters and not only the lowest energy trap contribute to the low temperature emission spectrum. The average emission spectrum obtained from more than hundred single complexes shows three main contributions that are in good agreement with the reported bands F685, F689 and F695. The intensity of F695 is found to be lower than in conventional ensemble spectroscopy. The reason for the deviation might be due to the accumulation of triplet states on the red-most chlorophylls (e.g. Chl29 in CP47) or on carotenoids close to these long-wavelength traps by the high excitation power used in the single-molecule experiments. The red-most emitter will not contribute to the fluorescence spectrum as long as it is in the triplet state. In addition, quenching of fluorescence by the triplet state may lead to a decrease of long-wavelength emission.
Asunto(s)
Complejo de Proteína del Fotosistema II/química , Espectrometría de Fluorescencia/métodos , Espectrofotometría Ultravioleta/métodos , Synechococcus/química , Dimerización , Modelos MolecularesRESUMEN
The oxygen-evolving complex (OEC) in the membrane-bound protein complex photosystem II (PSII) catalyzes the water oxidation reaction that takes place in oxygenic photosynthetic organisms. We investigated the structural changes of the Mn4CaO5 cluster in the OEC during the S state transitions using x-ray absorption spectroscopy (XAS). Overall structural changes of the Mn4CaO5 cluster, based on the manganese ligand and Mn-Mn distances obtained from this study, were incorporated into the geometry of the Mn4CaO5 cluster in the OEC obtained from a polarized XAS model and the 1.9-Å high resolution crystal structure. Additionally, we compared the S1 state XAS of the dimeric and monomeric form of PSII from Thermosynechococcus elongatus and spinach PSII. Although the basic structures of the OEC are the same for T. elongatus PSII and spinach PSII, minor electronic structural differences that affect the manganese K-edge XAS between T. elongatus PSII and spinach PSII are found and may originate from differences in the second sphere ligand atom geometry.
Asunto(s)
Oxígeno/química , Complejo de Proteína del Fotosistema II/química , CatálisisRESUMEN
Intense femtosecond x-ray pulses produced at the Linac Coherent Light Source (LCLS) were used for simultaneous x-ray diffraction (XRD) and x-ray emission spectroscopy (XES) of microcrystals of photosystem II (PS II) at room temperature. This method probes the overall protein structure and the electronic structure of the Mn4CaO5 cluster in the oxygen-evolving complex of PS II. XRD data are presented from both the dark state (S1) and the first illuminated state (S2) of PS II. Our simultaneous XRD-XES study shows that the PS II crystals are intact during our measurements at the LCLS, not only with respect to the structure of PS II, but also with regard to the electronic structure of the highly radiation-sensitive Mn4CaO5 cluster, opening new directions for future dynamics studies.
Asunto(s)
Compuestos de Manganeso/química , Óxidos/química , Complejo de Proteína del Fotosistema II/química , Cristalografía por Rayos X/métodos , Cianobacterias/enzimología , Electrones , Luz , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/efectos de la radiación , Conformación Proteica , Espectrometría por Rayos X/métodos , Temperatura , Agua/química , Difracción de Rayos X/métodosRESUMEN
The ultrabright femtosecond X-ray pulses provided by X-ray free-electron lasers open capabilities for studying the structure and dynamics of a wide variety of systems beyond what is possible with synchrotron sources. Recently, this "probe-before-destroy" approach has been demonstrated for atomic structure determination by serial X-ray diffraction of microcrystals. There has been the question whether a similar approach can be extended to probe the local electronic structure by X-ray spectroscopy. To address this, we have carried out femtosecond X-ray emission spectroscopy (XES) at the Linac Coherent Light Source using redox-active Mn complexes. XES probes the charge and spin states as well as the ligand environment, critical for understanding the functional role of redox-active metal sites. Kß(1,3) XES spectra of Mn(II) and Mn(2)(III,IV) complexes at room temperature were collected using a wavelength dispersive spectrometer and femtosecond X-ray pulses with an individual dose of up to >100 MGy. The spectra were found in agreement with undamaged spectra collected at low dose using synchrotron radiation. Our results demonstrate that the intact electronic structure of redox active transition metal compounds in different oxidation states can be characterized with this shot-by-shot method. This opens the door for studying the chemical dynamics of metal catalytic sites by following reactions under functional conditions. The technique can be combined with X-ray diffraction to simultaneously obtain the geometric structure of the overall protein and the local chemistry of active metal sites and is expected to prove valuable for understanding the mechanism of important metalloproteins, such as photosystem II.
RESUMEN
An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14-3.1 µl min(-1) to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min(-1) and diffracted to beyond 4 Å resolution, producing 14,000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.
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Cristalografía por Rayos X/instrumentación , Cristalización , Cristalografía por Rayos X/economía , Campos Electromagnéticos , Diseño de Equipo , Cinética , Rayos Láser , Tamaño de la Muestra , Termolisina/químicaRESUMEN
Most of the dioxygen on earth is generated by the oxidation of water by photosystem II (PS II) using light from the sun. This light-driven, four-photon reaction is catalyzed by the Mn(4)CaO(5) cluster located at the lumenal side of PS II. Various X-ray studies have been carried out at cryogenic temperatures to understand the intermediate steps involved in the water oxidation mechanism. However, the necessity for collecting data at room temperature, especially for studying the transient steps during the O-O bond formation, requires the development of new methodologies. In this paper we report room temperature X-ray diffraction data of PS II microcrystals obtained using ultrashort (< 50 fs) 9 keV X-ray pulses from a hard X-ray free electron laser, namely the Linac Coherent Light Source. The results presented here demonstrate that the "probe before destroy" approach using an X-ray free electron laser works even for the highly-sensitive Mn(4)CaO(5) cluster in PS II at room temperature. We show that these data are comparable to those obtained in synchrotron radiation studies as seen by the similarities in the overall structure of the helices, the protein subunits and the location of the various cofactors. This work is, therefore, an important step toward future studies for resolving the structure of the Mn(4)CaO(5) cluster without any damage at room temperature, and of the reaction intermediates of PS II during O-O bond formation.
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Cristalografía por Rayos X/métodos , Complejo de Proteína del Fotosistema II/química , Catálisis , Cristalización , Modelos MolecularesRESUMEN
High-pressure freezing (HPF) is a method which allows sample vitrification without cryoprotectants. In the present work, protein crystals were cooled to cryogenic temperatures at a pressure of 210 MPa. In contrast to other HPF methods published to date in the field of cryocrystallography, this protocol involves rapid sample cooling using a standard HPF device. The fast cooling rates allow HPF of protein crystals directly in their mother liquor without the need for cryoprotectants or external reagents. HPF was first attempted with hen egg-white lysozyme and cubic insulin crystals, yielding good to excellent diffraction quality. Non-cryoprotected crystals of the membrane protein photosystem II have been successfully cryocooled for the first time. This indicates that the presented HPF method is well suited to the vitrification of challenging systems with large unit cells and weak crystal contacts.
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Cristalografía por Rayos X/métodos , Insulina/análisis , Muramidasa/análisis , Animales , Pollos , Cristalografía por Rayos X/instrumentación , Congelación , Presión , Factores de TiempoRESUMEN
The photosystem II core complex is the water:plastoquinone oxidoreductase of oxygenic photosynthesis situated in the thylakoid membrane of cyanobacteria, algae and plants. It catalyzes the light-induced transfer of electrons from water to plastoquinone accompanied by the net transport of protons from the cytoplasm (stroma) to the lumen, the production of molecular oxygen and the release of plastoquinol into the membrane phase. In this review, we outline our present knowledge about the "acceptor side" of the photosystem II core complex covering the reaction center with focus on the primary (Q(A)) and secondary (Q(B)) quinones situated around the non-heme iron with bound (bi)carbonate and a comparison with the reaction center of purple bacteria. Related topics addressed are quinone diffusion channels for plastoquinone/plastoquinol exchange, the newly discovered third quinone Q(C), the relevance of lipids, the interactions of quinones with the still enigmatic cytochrome b559 and the role of Q(A) in photoinhibition and photoprotection mechanisms. This article is part of a Special Issue entitled: Photosystem II.
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Luz , Complejo de Proteína del Fotosistema II/metabolismo , Quinonas/metabolismo , Transferencia de Energía , Modelos Moleculares , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/químicaRESUMEN
PURPOSE: Despite their excellent clinical validity, objective measures of memory often do not reflect self-perceived memory impairment. This discordance has mostly been attributed to depressed mood. Alternatively, a lack of ecological validity due to the rather short standard retention intervals of 20-60 min may be responsible for this discordance. Therefore, we explored the value of extended retention intervals in regard to subjective memory deficits. METHODS: Our prospective study was based on 73 patients with epilepsy. In addition to the standard 30-min retention interval of a verbal learning and memory test (VLMT) patients were randomized to either a free delayed recall after 1 week or after 4 weeks. Mood was assessed by the Beck Depression Inventory (BDI). RESULTS: Forty-four patients (60%) reported self-perceived memory deficits, whereas objective verbal memory impairment was present in 26 patients (36%). Concordance between subjective and objective memory performance was observed in 53% of the patients. Multivariate analyses identified memory performance after 4 weeks and self-rated mood as determinants of subjective memory impairment. Self-perceived memory impairment correlated with the number of remembered words after 4 weeks (r = -0.361, p = 0.030) and the BDI total score (r = 0.332, p = 0.004) but neither with recall performance after 30 min nor after 1 week. CONCLUSION: Subjective memory appears to follow a different time scale than routine memory testing. Thus, the introduction of longer retention intervals may enhance the ecological validity of standard memory tests. Furthermore, the findings again underscore that controlling for mood is mandatory when dealing with subjective memory complaints.
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Epilepsia/fisiopatología , Trastornos de la Memoria/diagnóstico , Recuerdo Mental/fisiología , Retención en Psicología/fisiología , Adulto , Afecto/fisiología , Anciano , Femenino , Humanos , Masculino , Trastornos de la Memoria/etiología , Persona de Mediana Edad , Pruebas Neuropsicológicas , Aprendizaje Verbal , Adulto JovenRESUMEN
Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the Q(B) site in the D1 subunit and thus block the electron transfer from Q(A) to Q(B). Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å. The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the Q(B) site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of Q(A), which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334-342). This discovery is discussed in the context of proton transfer to the lumen.
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Cianobacterias/enzimología , Herbicidas/química , Complejo de Proteína del Fotosistema II/química , Triazinas/química , Cristalografía por Rayos X , Herbicidas/farmacología , Complejo de Proteína del Fotosistema II/antagonistas & inhibidores , Complejo de Proteína del Fotosistema II/metabolismo , Estructura Cuaternaria de Proteína , Relación Estructura-Actividad , Triazinas/farmacologíaRESUMEN
The photosynthetic oxygen-evolving photosystem II (PSII) is the only known biochemical system that is able to oxidize water molecules and thereby generates almost all oxygen in the Earth's atmosphere. The elucidation of the structural and mechanistic aspects of PSII keeps scientists all over the world engaged since several decades. In this Minireview, we outline the progress in understanding PSII based on the most recent crystal structure at 2.9 A resolution. A likely position of the chloride ion, which is known to be required for the fast turnover of water oxidation, could be determined in native PSII and is compared with work on bromide and iodide substituted PSII. Moreover, eleven new integral lipids could be assigned, emphasizing the importance of lipids for the perfect function of PSII. A third plastoquinone molecule (Q(C)) and a second quinone transfer channel are revealed, making it possible to consider different mechanisms for the exchange of plastoquinone/plastoquinol molecules. In addition, possible transport channels for water, dioxygen and protons are identified.