Alignment and Test of the Wide Field Infrared Survey Telescope (WFIRST) Engineering Design Unit (EDU) Grism

The WFIRST wide field instrument (WFI) includes a slitless spectrometer, which plays an important role in the WFIRST mission for the survey of emission-line galaxies. WFI is building engineering design and environmental test (EDU and ETU) units to reduce risk for the flight grism unit. We report here on successful build and test of the EDU grism. The four-element EDU grism consists of two prism elements and two diffractive elements that provide R700 dispersion. The elements were fabricated with alignment fiducials and integral flats to allow opto-mechanical alignment in six-degrees of freedom. Each element in turn, was installed onto a hexapod and positioned to its nominal orientation relative to the grism deck, then bonded into individual cells. Alignment measurements were performed in situ using theodolites to set tip/tilt and a Micro-vu non-contact Multisensor Measurement System was used to set despace, decenter and clocking of each element using the hexapod. After opto-mechanical alignment, the grism wavefront was measured using an Infrared ZYGO interferometer at various field points extending over a 20 by 14- degree (local) field of view. Using modeled alignment sensitivities, we determined the alignment correction required on our Element 2 prism compensator and successfully minimized the field dependent wavefront error and confocality. This paper details the alignment and testing of the EDU grism at ambient and cold operating temperatures

WFIRST Grism Characterization

We had presented the optical design and early test result of WFIRST grism spectrometer in previous SPIE conferences. This paper reports the follow-on activity of the spectral and radiometric calibrations, including the calibration methods, experiment designs, and test equipment calibration, such as the light source and detectors used in the test. The real grism calibration includes the throughput versus wavelength, which is largely determined by the diffraction efficiency of the two diffractive surfaces. It also includes spectral resolution, point spread function, and encircle energy measurements. The measured data are presented. The comparisons between the test data and the simulation from theory or optical model are also presented

Structural study of the membrane protein MscL using cell-free expression and solid-state

a b s t r a c t High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric a-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR

Etude de canaux ioniques de Escherichia coli par la methoe du patch clamp

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : TD 82119 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Méthodes de production et étude électrophysiologique de canaux ioniques (application à la pannexine1 humaine et au canal mécanosensible bactérien MscL)

La production hétérologue des protéines membranaires reste difficile, peut-être parce que l insertion dans la membrane de la cellule hôte constitue une étape limitante de la production. Afin de tourner cette difficulté, deux modes de synthèse ont été envisagés: la synthèse de protéines dans un système a-cellulaire, en l absence de membrane mais en présence de détergent, ou l adressage forcé de la protéine vers les corps d inclusion dans le cas d une expression plus classique en bactérie entière. La réalisation des deux stratégies repose sur l utilisation de protéines de fusion possédant une séquence d entraînement en amont du gène d intérêt, soit qu elles améliorent la traduction du transcrit en limitant le repliement spatial de ce dernier, soit qu elles favorisent la production de la protéine d intérêt en corps d inclusion. La porine OmpX et le peptide T7 ont été choisis en cas d expression dans les systèmes bactériens. La protéine SUMO est utilisée pour la production dans un lysat eucaryote. Les différentes approches ont été testées sur la production de la pannexine1 humaine (Px1).Si les séquences d entraînement OmpX et le peptide T7 sont correctement produites in vitro, aucune des deux, en revanche, ne favorise la production de la Px1. Seul l entraîneur SUMO est efficace. En effet, nous avons observé que cette protéine augmente la production de la Px1 dans un lysat eucaryote de germe de blé. Par ailleurs OmpX, connue pour être largement produite in vivo dans les corps d inclusion, n entraîne pas la localisation de la Px1 dans ces structures. Contre toute attente, l étiquette T7 dirige la Px1 dans les corps d inclusion. L étude électrophysiologique de la Px1 a donc été effectuée à partir de la protéine produite in vivo (T7his-Px1) après renaturation, ou produite sous forme soluble in vitro (his6-Px1) dans le lysat eucaryote. Dans le cas de la protéine T7his-Px1 renaturée, une activité canal qui rappelle celle qui est observée après expression dans l ovocyte de Xénope, a été détectée en patch-clamp, mais dans trois cas seulement. Dans le cas de la protéine his6-Px1, aucune activité canal n est clairement détectée. Dans une deuxième partie de ce travail on examine le rôle de la boucle périplasmique dans la sensibilité à la pression du MscL, un canal mécanosensible bactérien devenu un système modèle dans l étude de la mécanosensibilité. Presque toutes les études fonctionnelles sur ce canal ont été réalisées sur le canal de E.coli, alors que la structure a été obtenue à partir de l homologue de M. tuberculosis. Une étude fonctionnelle a montré que le MscL de M. tuberculosis est difficile à ouvrir : son ouverture requiert l application d une pression double de celle qui est nécessaire chez E.coli. Les deux homologues diffèrent principalement par la longueur de leur unique boucle périplasmique. De manière à examiner le rôle de la boucle, on a comparé l activité du canal MscL de E.coli, celle du canal de M. tuberculosis et celle d une protéine chimère constituée de la protéine de M. tuberculosis dans laquelle la boucle a été changée pour celle de la protéine de E.coli. De manière inattendue, nous avons constaté que les canaux de E.coli et de M. tuberculosis ont la même sensibilité à la pression. La protéine chimère n avait pas d activité canal. Si ce travail ne permet pas de conclure quant au rôle de la boucle, il montre sans ambigüité que contrairement à ce qui a été rapporté les canaux MscL de E.coli et de M. tuberculosis ne diffèrent pas sensiblement sur le plan fonctionnelThe production of heterologous membrane protein is notoriously difficult; this might be due to the fact that insertion of the protein in the membrane host is a limiting step. To by-pass this difficulty, two modes of synthesis were tested: 1) production in a cell-free system devoid of biological membrane but supplemented with detergent or liposomes, 2) production in bacteria, with targeting of the membrane protein to inclusion bodies. Both strategies were tested for the production of the human pannexin 1 channel (Px1). The gene coding the protein was fused with an enhancer sequence resulting in the addition of a peptide or short protein at the N terminus of the protein of interest. This enhancer sequence which is well produced in vitro or in vivo is supposed to facilitate the translation of the protein of interest. Three enhancer sequences were chosen: 1) the small porin OmpX of E. coli, which, in addition, should target the protein to inclusion bodies when the protein is expressed in bacteria 2) a peptide of phage T7 for expression in E.coli lysate or E.coli cells 3) the small protein SUMO for production in a wheat germ cell-free system. In a bacterial cell-free system, neither OmpX nor T7 promoted Px1 production. Px1 is only produced when the SUMO enhancer sequence is used in the wheat germ system. In bacteria, OmpX, known to form inclusions bodies did not promote the targeting of the fusion protein to inclusion bodies. Unexpectedly, the peptide T7 was able to do it.Px1 obtained from inclusion bodies (T7his-Px1) was renatured and reconstituted in liposomes. Similarly his6-Px1 produced in wheat germ system was reconstituted in liposomes. Both preparations were used for electrophysiological studies (patch-clamp and planar bilayers). With the refolded T7his-Px1, channel activity reminiscent of that observed with Px1 expressed in Xenope oocyte (Bao et al., 2004) could be detected, but only in three cases. In the case of his6-Px1, no clear channel activity could be observed. The second part of this work deals with the involvement of the periplasmic loop of the bacterial mechanosensitive channel MscL in its sensitivity to pressure. Mscl has become a model system for the investigation of mechanosensisity. Nearly all functional studies have been performed on MscL from E.coli while the structure of the protein has been obtained from the Mycobacterium tuberculosis homologue. In one functional study it was shown that MscL from M. tuberculosis is extremely difficult to open, gating at twice the pressure needed for E.coli MscL The periplasmic loop is the most variable sequence between the two homologues, being longer in E.coli than in M. tuberculosis. In order to assess the role of the periplamic loop in the sensitivity to pressure, we compared the activity of the E.coli and M. tuberculosis MscL and of a chimeric protein made of the M. tuberculosis protein in which the periplasmic loop has been exchanged for that of the E. coli channel. Unexpectedly, M. tuberculosis and E .coli MscL were observed to gate at a similar applied pressure. The chimeric protein had no functional activity. In conclusion, this study does not allow any conclusion as to the role of the loop in the sensitivity to pressure, but it shows clearly that, in contrast to the results of a previous study, there is no functional difference between E. coli and M. tuberculosis MscL.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Function and Expression of an N-Acetylneuraminic Acid-Inducible Outer Membrane Channel in Escherichia coli

The Escherichia coli yjhA (renamed nanC) gene encodes a protein of the KdgM family of outer membrane-specific channels. It is transcribed divergently from fimB, a gene involved in the site-specific inversion of the region controlling transcription of the fimbrial structural genes but is separated from it by one of the largest intergenic regions in E. coli. We show that nanC expression is induced by N-acetylneuraminic acid and modulated by N-acetylglucosamine. This regulation occurs via the NanR and NagC regulators, which also control fimB expression. nanC expression is also activated by the regulators cyclic AMP-catabolite activator protein, OmpR, and CpxR. When the NanC protein was reconstituted into liposomes, it formed channels with a conductance of 450 pS at positive potential and 300 to 400 pS at negative potential in 800 mM KCl. The channels had a weak anionic selectivity. In an ompR background, where the general porins OmpF and OmpC are absent, NanC is required for growth of E. coli on N-acetylneuraminic acid as the sole carbon source. All these results suggest that NanC is an N-acetylneuraminic acid outer membrane channel protein

Purification and Functional Reconstitution of N- and C-Halves of the MscL Channel

MscL is a mechanosensitive channel gated by membrane tension in the lipid bilayer alone. Its structure, known from x-ray crystallography, indicates that it is a homopentamer. Each subunit comprises two transmembrane segments TM1 and TM2 connected by a periplasmic loop. The closed pore is lined by five TM1 helices. We expressed in Escherichia coli and purified two halves of the protein, each containing one of the transmembrane segments. Their electrophysiological activity was studied by the patch-clamp recording upon reconstitution in artificial liposomes. The TM2 moiety had no electrophysiological activity, whereas the TM1 half formed channels, which were not affected by membrane tension and varied in conductance between 50 and 350 pS in 100 mM KCl. Coreconstitution of the two halves of MscL however, yielded mechanosensitive channels having the same conductance as the native MscL (1500 pS), but exhibiting increased sensitivity to pressure. Our results confirm the current view on the functional role of TM1 and TM2 helices in the MscL gating and emphasize the importance of helix-helix interactions for the assembly and functional properties of the channel protein. In addition, the results indicate a crucial role of the periplasmic loop for the channel mechanosensitivity

The Oligogalacturonate-specific Porin KdgM of Erwinia chrysanthemi Belongs to a New Porin Family

International audienceThe phytopathogenic Gram-negative bacteria Erwinia chrysanthemi secretes pectinases, which are able to degrade the pectic polymers of plant cell walls, and uses the degradation products as a carbon source for growth. We characterized a major outer membrane protein, KdgM, whose synthesis is strongly induced in the presence of pectic derivatives. The corresponding gene was characterized. Analysis of transcriptional fusions showed that the kdgM expression is controlled by the general repres-sor of pectinolytic genes, KdgR, by the repressor of hexu-ronate catabolism genes, ExuR, by the pectinase gene repressor, PecS, and by catabolite repression via the cyclic AMP receptor protein (CRP) transcriptional activa-tor. A kdgM mutant is unable to grow on oligogalactu-ronides longer than trimers, and its virulence is affected. Electrophysiological experiments with planar lipid bilay-ers showed that KdgM behaves like a voltage-dependent porin that is slightly selective for anions and that exhibits fast block in the presence of trigalacturonate. In contrast to most porins, KdgM seems to be monomeric. KdgM has no homology with currently known porins, but proteins similar to KdgM are present in several bacteria. Therefore , these proteins might constitute a new family of porin channels