RESUMO
Event-based clustering provides a low-power embedded solution for low-level feature extraction in a scene. The algorithm utilizes the non-uniform sampling capability of event-based image sensors to measure local intensity variations within a scene. Consequently, the clustering algorithm forms similar event groups while simultaneously estimating their attributes. This work proposes taking advantage of additional event information in order to provide new attributes for further processing. We elaborate on the estimation of the object velocity using the mean motion of the cluster. Next, we are examining a novel form of events, which includes intensity measurement of the color at the concerned pixel. These events may be processed to estimate the rough color of a cluster, or the color distribution in a cluster. Lastly, this paper presents some applications that utilize these features. The resulting algorithms are applied and exercised thanks to a custom event-based simulator, which generates videos of outdoor scenes. The velocity estimation methods provide satisfactory results with a trade-off between accuracy and convergence speed. Regarding color estimation, the luminance estimation is challenging in the test cases, while the chrominance is precisely estimated. The estimated quantities are adequate for accurately classifying objects into predefined categories.
RESUMO
Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.
Assuntos
Cianobactérias/química , Elétrons , Lasers , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Temperatura , Amônia/química , Amônia/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalização , Manganês/metabolismo , Modelos Moleculares , Oxigênio/metabolismo , Especificidade por Substrato , Água/metabolismoRESUMO
Suffering is a universal entity, multidimensional, but also unique and personal. Unfortunately, it is often underdiagnosed, while it is omnipresent in our hospital practice. This article offers to physicians some ideas for the exploration and identification of the suffering of an end of life patient's relatives, and especially some tools for improving ways to support.
La souffrance est une entité universelle, multidimensionnelle, mais aussi unique et personnelle, paradoxalement sous-diagnostiquée, alors qu'elle est omniprésente dans notre pratique en milieu hospitalier. Le but de cet article est de proposer au lecteur quelques pistes pour l'exploration et l'identification de la souffrance des proches de patients en situation palliative, et surtout quelques outils d'accompagnement et de soutien.
Assuntos
Médicos , Assistência Terminal , Hospitais , HumanosRESUMO
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.
Assuntos
Lasers , Substâncias Macromoleculares/química , Bacillus/enzimologia , Cálcio/química , Calibragem , Simulação por Computador , Cristalização , Cristalografia por Raios X , Elétrons , Desenho de Equipamento , Funções Verossimilhança , Modelos Químicos , Conformação Molecular , Muramidase/química , Nanotecnologia , Reprodutibilidade dos Testes , Software , Termolisina/química , Raios X , Zinco/químicaRESUMO
Well-defined mixed-metal [CoMn3 O4 ] and [NiMn3 O4 ] cubane complexes were synthesized and used as precursors for heterogeneous oxygen evolution reaction (OER) electrocatalysts. The discrete clusters were dropcasted onto glassy carbon (GC) and indium tin oxide (ITO) electrodes, and the OER activities of the resulting films were evaluated. The catalytic surfaces were analyzed by various techniques to gain insight into the structure-function relationships of the electrocatalysts' heterometallic composition. Depending on preparation conditions, the Co-Mn oxide was found to change metal composition during catalysis, while the Ni-Mn oxides maintained the NiMn3 ratio. XAS studies provided structural insights indicating that the electrocatalysts are different from the molecular precursors, but that the original NiMn3 O4 cubane-like geometry was maintained in the absence of thermal treatment (2-Ni). In contrast, the thermally generated 3-Ni develops an oxide-like extended structure. Both 2-Ni and 3-Ni undergo structural changes upon electrolysis, but they do not convert into the same material. The observed structural motifs in these heterogeneous electrocatalysts are reminiscent of the biological oxygen-evolving complex in Photosystemâ II, including the MMn3 O4 cubane moiety. The reported studies demonstrate the use of discrete heterometallic oxide clusters as precursors for heterogeneous water oxidation catalysts of novel composition and the distinct behavior of two sets of mixed metal oxides.
Assuntos
Compostos de Manganês/química , Metais/química , Óxidos/química , Oxigênio/química , Catálise , Eletrodos , Íons/química , OxirreduçãoRESUMO
Herein, Ca K-edge X-ray absorption spectroscopy (XAS) is developed as a means to characterize the local environment of calcium centers. The spectra for six, seven, and eight coordinate inorganic and molecular calcium complexes were analyzed and determined to be primarily influenced by the coordination environment and site symmetry at the calcium center. The experimental results are closely correlated to time-dependent density functional theory (TD-DFT) calculations of the XAS spectra. The applicability of this methodology to complex systems was investigated using structural mimics of the oxygen-evolving complex (OEC) of PSII. It was found that Ca K-edge XAS is a sensitive probe for structural changes occurring in the cubane heterometallic cluster due to Mn oxidation. Future applications to the OEC are discussed.
Assuntos
Cálcio/química , Manganês/química , Compostos Organometálicos/química , Compostos Organometálicos/síntese química , Teoria Quântica , Espectroscopia por Absorção de Raios XRESUMO
Tracing the transient atomic motions that lie at the heart of chemical reactions requires high-resolution multidimensional structural information on the timescale of molecular vibrations, which commonly range from 10 fs to 1 ps. For simple chemical systems, it has been possible to map out in considerable detail the reactive potential-energy surfaces describing atomic motions and resultant reaction dynamics, but such studies remain challenging for complex chemical and biological transformations. A case in point is the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, which is a widely used gene expression marker owing to its efficient bioluminescence. This feature is known to arise from excited-state proton transfer (ESPT), yet the atomistic details of the process are still not fully understood. Here we show that femtosecond stimulated Raman spectroscopy provides sufficiently detailed and time-resolved vibrational spectra of the electronically excited chromophore of GFP to reveal skeletal motions involved in the proton transfer that produces the fluorescent form of the protein. In particular, we observe that the frequencies and intensities of two marker bands, the C-O and C = N stretching modes at opposite ends of the conjugated chromophore, oscillate out of phase with a period of 280 fs; we attribute these oscillations to impulsively excited low-frequency phenoxyl-ring motions, which optimize the geometry of the chromophore for ESPT. Our findings illustrate that femtosecond simulated Raman spectroscopy is a powerful approach to revealing the real-time nuclear dynamics that make up a multidimensional polyatomic reaction coordinate.
Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Vibração , Animais , Evolução Molecular , Proteínas de Fluorescência Verde/genética , Modelos Moleculares , Movimento , Prótons , Análise Espectral Raman , Fatores de TempoRESUMO
Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.
Assuntos
Citocromos c/metabolismo , Bactérias Gram-Positivas/metabolismo , Heme/metabolismo , Metais/metabolismo , Peptococcaceae/enzimologia , Cromatografia Líquida , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão , Oxirredução , Peptococcaceae/ultraestrutura , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Cristalografia por Raios X/métodos , Complexo de Proteína do Fotossistema II/química , Catálise , Cristalização , Modelos MolecularesRESUMO
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.
RESUMO
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.
Assuntos
Cristalografia por Raios X/instrumentação , Cristalização , Cristalografia por Raios X/economia , Campos Eletromagnéticos , Desenho de Equipamento , Cinética , Lasers , Tamanho da Amostra , Termolisina/químicaRESUMO
Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O(2))-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket. The mutations significantly increased O(2) association rates (1.5-2.5-fold) and dissociation rates (8-190-fold) as compared to wild-type Tt H-NOX. Structural changes that accompanied the I5F mutation were characterized using X-ray crystallography and resonance Raman spectroscopy. A 1.67 Å crystal structure of the I5F mutant indicated that introducing a phenylalanine at position 5 resulted in a significant shift of the N-terminal domain of the protein, causing an opening of the heme pocket. This movement also resulted in an increased amount of flexibility at the N-terminus and the loop covering the N-terminal helix as indicated by the two conformations of the first six N-terminal amino acids, high B-factors in this region of the protein, and partially discontinuous electron density. In addition, introduction of a phenylalanine at position 5 resulted in increased flexibility of the heme within the pocket and weakened hydrogen bonding to the bound O(2) as measured by resonance Raman spectroscopy. This study provides insight into the critical role of I5 in controlling conformational flexibility and ligand affinity in H-NOX proteins.
Assuntos
Hemeproteínas/metabolismo , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Cristalografia por Raios X , Hemeproteínas/química , Modelos Moleculares , Óxido Nítrico/química , Conformação Proteica , Espectrofotometria Ultravioleta , Análise Espectral Raman , Thermoanaerobacter/metabolismoRESUMO
The Heme Nitric oxide/OXygen binding (H-NOX) family of proteins have important functions in gaseous ligand signaling in organisms from bacteria to humans, including nitric oxide (NO) sensing in mammals, and provide a model system for probing ligand selectivity in hemoproteins. A unique vibrational feature that is ubiquitous throughout the H-NOX family is the presence of a high C-O stretching frequency. To investigate the cause of this spectroscopic characteristic, the Fe-CO and C-O stretching frequencies were probed in the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) using resonance Raman (RR) spectroscopy. Four classes of heme pocket mutants were generated to assess the changes in stretching frequency: (i) the distal H-bonding network, (ii) the proximal histidine ligand, (iii) modulation of the heme conformation via Ile-5 and Pro-115, and (iv) the conserved Tyr-Ser-Arg (YxSxR) motif. These mutations revealed important electrostatic interactions that dampen the back-donation of the Fe(II) d(π) electrons into the CO π* orbitals. The most significant change occurred upon disruption of the H-bonds between the strictly conserved YxSxR motif and the heme propionate groups, producing two dominant CO-bound heme conformations. One conformer was structurally similar to Tt H-NOX WT, whereas the other displayed a decrease in ν(C-O) of up to â¼70 cm(-1) relative to the WT protein, with minimal changes in ν(Fe-CO). Taken together, these results show that the electrostatic interactions in the Tt H-NOX binding pocket are primarily responsible for the high ν(C-O) by decreasing the Fe d(π) â CO π* back-donation and suggest that the dominant mechanism by which this family modulates the Fe(II)-CO bond likely involves the YxSxR motif.
Assuntos
Proteínas de Bactérias/química , Monóxido de Carbono/química , Hemeproteínas/química , Óxido Nítrico/química , Oxigênio/química , Thermoanaerobacter/química , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Compostos Ferrosos/química , Hemeproteínas/genética , Humanos , Ligação de Hidrogênio , Ligantes , Mutagênese Sítio-Dirigida , Óxido Nítrico/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Transdução de Sinais/genética , Análise Espectral Raman , Eletricidade Estática , Thermoanaerobacter/genética , VibraçãoRESUMO
Globins are heme-containing proteins that are best known for their roles in oxygen (O(2)) transport and storage. However, more diverse roles of globins in biology are being revealed, including gas and redox sensing. In the nematode Caenorhabditis elegans, 33 globin or globin-like genes were recently identified, some of which are known to be expressed in the sensory neurons of the worm and linked to O(2) sensing behavior. Here, we describe GLB-6, a novel globin-like protein expressed in the neurons of C. elegans. Recombinantly expressed full-length GLB-6 contains a heme site with spectral features that are similar to those of other bis-histidyl ligated globins, such as neuroglobin and cytoglobin. In contrast to these globins, however, ligands such as CO, NO, and CN(-) do not bind to the heme in GLB-6, demonstrating that the endogenous histidine ligands are likely very tightly coordinated. Additionally, GLB-6 exhibits rapid two-state autoxidation kinetics in the presence of physiological O(2) levels as well as a low redox potential (-193 +/- 2 mV). A high-resolution (1.40 A) crystal structure of the ferric form of the heme domain of GLB-6 confirms both the putative globin fold and bis-histidyl ligation and also demonstrates key structural features that can be correlated with the unusual ligand binding and redox properties exhibited by the full-length protein. Taken together, the biochemical properties of GLB-6 suggest that this neural protein would most likely serve as a physiological sensor for O(2) in C. elegans via redox signaling and/or electron transfer.
Assuntos
Caenorhabditis elegans/metabolismo , Globinas/química , Globinas/metabolismo , Animais , Citoglobina , Heme/química , Heme/metabolismo , Hemeproteínas/química , Hemeproteínas/metabolismo , Histidina , Cinética , Ligantes , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neuroglobina , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Células Receptoras SensoriaisRESUMO
Resonance Raman spectra were measured for the wild type Heme-Nitric oxide/OXygen binding domain from Thermoanaerobacter tengcongensis (Tt H-NOX WT) and three other Tt H-NOX proteins containing mutations at key conserved residues to determine the heme conformation in solution. The most dramatic changes in heme conformation occurred in the O2-bound forms, and the single Tt H-NOX P115A mutation was sufficient to generate a significant relaxation of the chromophore. Clear evidence of heme relaxation in the Tt H-NOX I5L, P115A, and I5L/P115A mutants in solution is demonstrated by the observation of reduced resonance Raman intensities for several out-of-plane low frequency modes (e.g., gamma11, gamma12, gamma13, and gamma15) in the 400-750 cm(-1) region known to be sensitive to ruffling and saddling deformations, as well as increased vibrational frequencies for the core heme skeletal stretching modes, nu3, nu2, and nu10. In addition, all three mutants exhibited some degree of heme conformational heterogeneity based on several broad skeletal markers (e.g., nu10) in the high frequency region. These results are comparable to those observed by Olea et al. for Tt H-NOX P115A in crystal form, where four different heme structures were determined from a single unit cell. On the basis of the resonance Raman spectra, it is clear that the actual heme conformation for Tt H-NOX P115A in solution is considerably more relaxed than that of the WT protein, with increased flexibility within the protein pocket, allowing for rapid sampling of alternate conformations.
Assuntos
Substituição de Aminoácidos/genética , Proteínas de Bactérias/química , Hemeproteínas/química , Óxido Nítrico/química , Oxigênio/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hemeproteínas/genética , Hemeproteínas/metabolismo , Isoleucina/genética , Ligantes , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Prolina/genética , Ligação Proteica/genética , Conformação Proteica , Estrutura Terciária de Proteína/genética , Análise Espectral Raman , ThermoanaerobacterRESUMO
It is known that when bilayers of some saturated phosphatidylcholines are stored for 3 or more days at approximately 0 degrees C, a lamellar subgel (Lc) phase is detected at temperatures below the pretransition by differential scanning calorimetry (DSC). However, the subgel (Lc) phase and the corresponding subtransition (Lc--> Lbeta') for dimyristoylphosphatidylcholine (DMPC) has not been clearly characterized. In this study, using the temperature jump protocol first developed by Tristram-Nagle et al. for the dipalmitoylphosphatidylcholine (DPPC) system, new and accurate data characterizing the subgel formation and subtransition of DMPC were obtained through DSC and fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene (DPH). It was discovered that the formation of the DMPC subgel phase requires incubation at temperatures of -5 degrees C or lower for 2 h or more. Kinetics of the subgel formation indicate that it is a very complex process and demonstrates that the planar gel phase is merely metastable below the subtransition, and not the thermodynamically stable phase. The subgel growth of DMPC is proven to be the dehydration of the headgroup region, and the subtransition is a process in which poorly hydrated DMPC becomes hydrated.
Assuntos
Bicamadas Lipídicas/química , Transição de Fase , 1,2-Dipalmitoilfosfatidilcolina , Dimiristoilfosfatidilcolina , Géis , Temperatura , ÁguaRESUMO
X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
Assuntos
Cristalografia por Raios X/instrumentação , Enzimas/química , Acústica , Cristalografia por Raios X/métodos , Modelos Moleculares , Muramidase/química , Conformação Proteica , Termolisina/químicaRESUMO
In photosynthesis, photosystem II (PSII) is the multi-subunit membrane protein complex that catalyzes photo-oxidation of water into dioxygen through the oxygen evolving complex (OEC). To understand the water oxidation reaction, it is important to get structural information about the transient and intermediate states of the OEC in the dimeric PSII core complex (dPSIIcc). In recent times, femtosecond X-ray pulses from the free electron laser (XFEL) are being used to obtain X-ray diffraction (XRD) data of dPSIIcc microcrystals at room temperature that are free of radiation damage. In our experiments at the XFEL, we used an electrospun liquid microjet setup that requires microcrystals less than 40 µm in size. In this study, we explored various microseeding techniques to get a high yield of monodisperse uniform-sized microcrystals. Monodisperse microcrystals of dPSIIcc of uniform size were a key to improve the stability of the jet and the quality of XRD data obtained at the XFEL. This was evident by an improvement of the quality of the datasets obtained, from 6.5Å, using crystals grown without the micro seeding approach, to 4.5Å using crystals generated with the new method.
RESUMO
Differential scanning calorimetry (DSC) and fluorescence spectroscopy are useful techniques for investigating the phase transitions of phospholipid bilayers. In this study, these methods have been extended to determine the effects of ethanol on DSPC and DSPC/2 mol.% cholesterol bilayers. The biphasic effect of the main transition was observed on the DSC heating scans above 0.60 M ethanol. In addition, the concentration at which the biphasic effect occurs is not significantly changed in the presence of 2 mol.% cholesterol. For the fluorescence studies, 1,6-diphenyl-1,3,5-hexatriene (DPH) has been incorporated into the bilayer to monitor the phase transitions through the displacement of DPH. This fluorescent probe is used to directly determine the onset of interdigitation in the bilayer systems as indicated by a large decrease in the DPH fluorescence intensity. The addition of cholesterol lowered and broadened the transition temperatures of the phosphatidylcholine (PC) system. However, 2 mol.% cholesterol did not have a significant effect on the induction of the interdigitated phase in DSPC as observed from the small difference in ethanol threshold concentration for the two systems. This suggests that DSPC forms a more stable interdigitated gel phase than other PCs with shorter acyl chains.
Assuntos
Colesterol/química , Etanol/química , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Varredura Diferencial de Calorimetria , Difenilexatrieno/química , Géis/química , Espectrometria de Fluorescência , TemperaturaRESUMO
The structure of photosystem II and the catalytic intermediate states of the Mn4CaO5 cluster involved in water oxidation have been studied intensively over the past several years. An understanding of the sequential chemistry of light absorption and the mechanism of water oxidation, however, requires a new approach beyond the conventional steady-state crystallography and X-ray spectroscopy at cryogenic temperatures. In this report, we present the preliminary progress using an X-ray free-electron laser to determine simultaneously the light-induced protein dynamics via crystallography and the local chemistry that occurs at the catalytic centre using X-ray spectroscopy under functional conditions at room temperature.