Lipidic cubic phase serial millisecond crystallography using synchrotron radiation.

Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins.Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven protonpump bacteriorhodopsin (bR) at a resolution of 2.4 A ° . The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein

Glutamate, GABA and Acetylcholine Signaling Components in the Lamina of the Drosophila Visual System

Synaptic connections of neurons in the Drosophila lamina, the most peripheral synaptic region of the visual system, have been comprehensively described. Although the lamina has been used extensively as a model for the development and plasticity of synaptic connections, the neurotransmitters in these circuits are still poorly known. Thus, to unravel possible neurotransmitter circuits in the lamina of Drosophila we combined Gal4 driven green fluorescent protein in specific lamina neurons with antisera to γ-aminobutyric acid (GABA), glutamic acid decarboxylase, a GABAB type of receptor, L-glutamate, a vesicular glutamate transporter (vGluT), ionotropic and metabotropic glutamate receptors, choline acetyltransferase and a vesicular acetylcholine transporter. We suggest that acetylcholine may be used as a neurotransmitter in both L4 monopolar neurons and a previously unreported type of wide-field tangential neuron (Cha-Tan). GABA is the likely transmitter of centrifugal neurons C2 and C3 and GABAB receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABAA receptor subunit RDL may be expressed by L4 neurons and a type of tangential neuron (rdl-Tan). Strong vGluT immunoreactivity was detected in α-processes of amacrine neurons and possibly in the large monopolar neurons L1 and L2. These neurons also express glutamate-like immunoreactivity. However, antisera to ionotropic and metabotropic glutamate receptors did not produce distinct immunosignals in the lamina. In summary, this paper describes novel features of two distinct types of tangential neurons in the Drosophila lamina and assigns putative neurotransmitters and some receptors to a few identified neuron types

Tolerance of triazine‐resistant and susceptible biotypes of three weeds to heat stress: a fluorescence study

The photosynthetic performance of Solarium nigrum L. Poa annua L. and Chenopodium album L. resistant to triazines was investigated in order to determine whether the alteration of the 32‐kD protein of photosystem (PS) II changed the ability to oxidize the PSII primary quinone acceptor QA. The effect of heat stress on the photochemistry of the resistant biotypes and the susceptible biotypes was also compared. The weeds were screened with the in‐vivo modulated chlorophyll fluorescence device to measure the photochemical component of fluorescence quenching (qQ), which provided semi‐quantitative information on the redox state of QA. At 25°C, an increase in the amplitude of the parameter 1‐qQ, which reflected the reduced state of QA, was observed in all resistant biotypes, compared to the susceptible wild biotypes. This was attributable to a shift in the equilibrium between QA− and QB (the PSII secondary quinone acceptor) in favour of QA. A heat stress of 35°C did not increase the level of reduced QA, except in the resistant biotypes of Poa annua. The photochemical activity of the two types of leaves exposed to increasingly high temperatures (25–65°C) indicated that quinone oxidation was more affected by heat stress in mutant resistant biotypes than in the susceptible biotypes. The quinone reoxidation was nullified at 60, 56 and 60°C, respectively, for susceptible biotypes of Solatium nigrum, Poa annua and Chenopodium album, and at 56, 48 and 54°C, respectively, for the three resistant biotypes. Heat also induced changes in the dark fluorescence F0, an indicator of the heat sensitivity of the light‐harvesting chlorophyll protein complex of PSII (LHCPII). The temperature dependence (25–70°C) of this fluorescence parameter confirmed the higher susceptibility of heat‐treated resistant leaves. Indeed, the temperatures of the peak of F0 (Tp) were 60, 55 and 62°C for susceptible Solanum nigrum, Poa annua and Chenopodium album, respectively. The Tp values for the three resistant biotypes were 55, 48 and 57°C, respectively. It is concluded that heat tolerance is related to differences in the organization of the chlorophyll antennae (LHCPII) between the two biotypes. Tolerance de biotypes sensibles et resistants aux triazines de 3 adventices au stress de la chaleur: une étude de fluorescence Les rendements photosynthétiques de Solanum nigrum L. Poa annua L. et Chenopodium album L. resistant aux triazines ont étéétudiés en vue de déterminer dans quelle mesure l'altération de la protéïne 32 kD du photosystème (PS) II, affecte la capacité d'oxyder l'accepteur quinonique primaire de PS II. QA L'influence d'un stress de chaleur sur la photochimie de biotypes résistants et de biotypes sensibles a été comparée. Les mauvaises herbes ont été triées au moyen de la fluorescence de la chlorophylle in vivo, pour mesurer la composante photochimique de réduction de la fluorescence (qQ) qui donne une information semi quantitative de l'état redox de QA. A 25 °C, une augmentation dans l'amplitude du paramètre 1‐qQ, qui reflète l'état réduit de QA a été observée chez les biotypes resistants encomparaison des biotypes sauvages sensibles. Ceci a été attribuéà une modification dans l'équilibre entre Q−A et QB (l'accepteur quinonique secondaire PS II), en faveur de QA. Un stress thermique de 35°C n'a pas augmenté le niveau de QA réduit, sauf chez le biotype résistant de Poa annua. L'activité photochimique des feuilles des 2 types exposées à des hautes températures croissantes (25 à 65°C) a montré que l'oxydation de la quinone était plus affectée par le stress thermique chez les biotypes mutants résistants que chez les biotypes sensibles. La réoxydation de la quinone était annulée à 60, 56 et 60°C respectivement pour les biotypes sensibles de Solatium nigrum, Poa annua et Chenopodium album et à 56, 48 et 54°C respectivement pour les 3 biotypes résistants. La chaleur a également entrainé des changements dans la fluorescence F0, un indicateur de la sensibilitéà la chaleur du complexe collecteur de lumière du PS II (LHCP II). La dépendance avec la température (25–70°C) de ce paramètre de fluorescence a confirmé la plus grande sensibilité des feuilles résistantes soumises à la chaleur. En effet, les températures du pic de Fo (Tp) étaient de 60, 55 et 62°C respectivement, pour les biotypes sensibles de Solanum nigrum, Poa annua et Chenopodium album. Les valeurs Tp pour les 3 biotypes résistants étaient respectivement de 55, 48 et 57°C. Il est conclu que la tolérance à la chaleur est liée aux différences dans l'organisation des antennes chlorophylliennes (LHC P II) entre les 2 biotypes. Hitzetoleranz Triazin‐resistenter und ‐empfindlicher Biotypen von drei Unkrautarten Die photosynthetische Leistung Triazin‐resistenter Biotypen von Solanum nigrum L. Poa annua L. und Chenopodium album L. wurde dahingehend untersucht, ob eine Veränderung des 32‐kD‐Eiweisses des Pigmentsystems II (PII) die Fähigkeit änderte, den primären Quinon‐Akzeptor QA des PII zu oxidieren. Auch die Wirkung einer Hitzebehandlung auf die photochemischen Reaktionen resistenter und empfindlicher Biotypen wurde verglichen. Die Unkräuter wurden in vivo mit einem Chlorophyll‐Fluorimeter gescreent, um die photochemische Komponente der Fluoreszenz‐Auslösung (qQ) zu messen, die eine semiquantitative Information über den Redox‐Zustand des QA lieferte. Die Amplitude des Parameters l‐qQ, der den reduzierten Zustand des QA widerspiegelte, nahm bei 25°C bei allen resistenten Biotypen im Vergleich zu den empfindlichen zu. Dies konnte einer Verschiebung des Gleichgewichts zwischen Q−A und QB (dem sekundären Quinon‐Akzeptor QA des PII) zugunsten des QA zugeordnet werden. Eine Hitzebehandlung mit 35°C erhöhte den reduzierten QA nicht, außer bei resistenten Biotypen von Poa annua. Die photochemische Aktivität von Blättern der beiden Typen, die steigenden Temperaturen von 25 bis 65°C unterworden wurden, zeigte, daß die Quinon‐Oxidation bei den resitenten Biotypen durch den Hitzestreß stärker beeinflußt wurde als bei den empfindlichen. Die Quinon‐Reoxidation wurde bei den empfindlichen Biotypen von Solanum nigrum, Poa annua und Chenopodium album bei 60, 56 bzw. 60°C aufgehoben, bei den resistenten bei 56, 48 bzw. 54°C. Hitze führte auch zu Änderungen der Dunkel‐Fluoreszenz Fo, einem Indikator der Hitzeempfindlichkeit des lichtabsorbierenden Chlorophyll‐Eiweißkomplexes des PII (LHCPII). Die Temperaturabhängigkeit dieses Fluoreszenz‐Parameters bei 25 bis 70°C bestätigte die höhere Empfindlichkeit hitzebehandelter Blätter resistenter Pflanzen. So waren Fo‐Peaks bei empfindlichen Solanum nigrum, Poa annua und Chenopodium album bei 60, 55 bzw. 62°C, bei resistenten bei 55, 48 bzw. 57°C festzustellen. Daraus wurde geschlossen, daß die Hitzetoleranz auf Unterschieden im Aufbau des lichtabsorbierenden Pigmentsystems LHCPII der beiden Biotypen beruht. Copyright © 1992, Wiley Blackwell. All rights reservedSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Femtosecond phase-transition in hard x-ray excited bismuth

The evolution of bismuth crystal structure upon excitation of its A(1g) phonon has been intensely studied with short pulse optical lasers. Here we present the first-time observation of a hard x-ray induced ultrafast phase transition in a bismuth single crystal at high intensities (similar to 10(14) W/cm(2)). The lattice evolution was followed using a recently demonstrated x-ray single-shot probing setup. The time evolution of the (111) Bragg peak intensity showed strong dependence on the excitation fluence. After exposure to a sufficiently intense x-ray pulse, the peak intensity dropped to zero within 300 fs, i.e. faster than one oscillation period of the A(1g) mode at room temperature. Our analysis indicates a nonthermal origin of a lattice disordering process, and excludes interpretations based on electron-ion equilibration process, or on thermodynamic heating process leading to plasma formation

The cAMP in thyroid: from the TSH receptor to mitogenesis and tumorigenesis

SCOPUS: re.jinfo:eu-repo/semantics/publishe

Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography

Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 angstrom resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX