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Accueil > Publications > Recherche par années > Années 2000 > 2000

2000

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A robust, detergent-friendly method for mass spectrometric analysis of integral membrane proteins

Recent breakthroughs in the high-resolution structural elucidation of ion channels and transporters are prompting a growing interest in methods for characterizing integral membrane proteins. These methods are proving extremely valuable in facilitating the production of X-ray diffraction-grade crystals. Here we present a robust and straightforward mass spectrometric procedure that utilizes matrix-assisted laser desorption/ionization to analyze integral membrane proteins in the presence of detergents. The utility of this method is illustrated with examples of high-quality mass spectral data obtained from membrane proteins for which atomic resolution structural studies are ongoing.

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A tertiary interaction that links active-site domains to the 5 ’ splice site of a group II intron

Group II introns are self-splicing RNAs that are commonly found in the genes of plants, fungi, yeast and bacteria(1,2). Little is known about the tertiary structure of group II introns, which are among the largest natural ribozymes. The most conserved region of the intron is domain 5 (D5), which, together with domain 1 (D1), is required for all reactions catalysed by the intron(3). Despite the importance of D5, its spatial relationship and tertiary contacts to other active-site constituents have remained obscure. Furthermore, D5 has never been placed directly at a site of catalysis by the intron. Here we show that a set of tertiary interactions (lambda-lambda’) links catalytically essential regions of D5 and D1, creating the framework for an active-site and anchoring it at the 5’ splice site. Highly conserved elements similar to components of the lambda-lambda’ interaction are found in the eukaryotic spliceosome.

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Action of a NO donor on the excitation-contraction pathway activated by noradrenaline in rat superior mesenteric artery

1. The aim of the present study was to investigate the actions of NO donors in rat superior mesenteric artery stimulated with noradrenaline by studying their effects on isometric tension, membrane potential (V-m), cytosolic calcium concentration ([Ca2+](cyt)) and accumulation of inositol phosphates.

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An ESA study for the search for life on Mars

Similarities in the early histories of Mars and Earth suggest the possibility that life may have arisen on Mars as it did on Earth. If this were the case, early deterioration of the environment on Mars (loss of surface water, decrease in temperature) may have inhibited further evolution of life. Thus, life on Mars would probably be similar to the simplest form of life on Earth, the prokaryotes. We present a hypothetical strategy to search for life on Mars consisting of (i) identifying a suitable landing site with good exobiological potential, and (ii) searching for morphological and biogeochemical signatures of extinct and extant life on the surface, in the regolith subsurface, and within rocks. The platform to be used in this theoretical exercise is an integrated, multi-user instrument package, distributed between a lander and rover, which will observe and analyse surface and subsurface samples to obtain the following information : 1. environmental data concerning the surface geology and mineralogy, UV radiation and oxidation processes ; 2. macroscopic to microscopic morphological evidence of life ; 3. biogeochemistry indicative of the presence of extinct or extant life ; 4. niches for extant life. Lastly, the rationale for human exploration of Mars will be addressed. (C) 2000 Elsevier Science Ltd. All rights reserved.

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Antibacterial and antifungal activities of vasostatin-1, the N-terminal fragment of chromogranin A

Vasostatin-1, the natural N-terminal 1-76 chromogranin A (CGA)-derived fragment in bovine sequence, has been purified from chromaffin secretory granules and identified by sequencing and matrix-assisted laser desorption time-of-flight mass spectrometry. This peptide, which displays antibacterial activity against Gram-positive bacteria at micromolar concentrations, is also able to kill a large variety of filamentous fungi and yeast cells in the 1-10 mu M range. We have found that the C-terminal moiety of vasostatin-1 is essential for the antifungal activity, and shorter active peptides have been synthesized.

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Catalytic and DNA binding properties of the Ogg1 protein of Saccharomyces cerevisiae : Comparison between the wild type and the K241R and K241Q active-site mutant proteins

The Ogg1 protein of Saccharomyces cerevisiae belongs to a family of DNA glycosylases and apurinic/apyrimidinic site (AP) lyases, the signature of which is the a-helix-hairpin-a-helix-Gly/Pro-Asp (HhH-GPD) active site motif together with a conserved catalytic lysine residue, to which we refer as the HhH-GPD/K family. In the yeast Ogg1 protein, yOgg1, the HhH-GPD/K motif spans residues 225-260 and the conserved lysine is K241. In this study, we have purified the K241R and K241Q mutant proteins and compared their catalytic and DNA binding properties to that of the wild-type yOgg1. The results show that the K241R mutation greatly impairs both the DNA glycosylase and the AP lyase activities of yOgg1.

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Changes in lac Operator dynamics upon selective interaction with lac Repressor as revealed by heteronuclear relaxation rate measurements

Structural and dynamic studies of the lac Operator complexed with the headpiece of the lac Repressor are necessary to establish whether the two partners have pre-required structures or if folding is essential to induce the specific interaction. NOESY spectra of the 1:1 complex of half of the lac Operator 5’d(CGCTCACAATT)-5’d(AATGTGAGCG) sequence (selectively labelled with C-13 at C-1’), with the Inc Repressor N-terminal headpiece 1-51 yielded information about the contact points between the nucleic acid and the protein. The operator was selectively labelled with C-13 at the C-1’ position, allowing the measurement of C-13 relaxation rates as a probe of dynamic behaviour.

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Changes of the potassium currents in rat aortic smooth muscle cells during postnatal development

This study was designed to investigate the role and regulation of arterial K+ channels during postnatal development. Rat thoracic aortic segments were suspended for isometric tension and resting membrane potential (RMP) recording. Contraction in response to 4-aminopyridine (4-AP) was similar in 4-, 8- and 12-week-old rats but was higher in 1-day-old rats. Contraction in response to tetraethylammonium (TEA) increased after 4 weeks. TEA increased the contractions evoked by noradrenaline in the aorta from 8- and 12-week-old rats but not from 1-day- and 4-week-old rats. RMP did not change during development. Patch-clamp studies of freshly isolated smooth muscle cells from the same aortas bathed in Ca2+-free medium showed a voltage-dependent Kf current (IK) sensitive to 4-AP.

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