Reconstitution of the Neurospora crassa plasma membrane H+-adenosine triphosphatase

AbstractThe purified H+-ATPase of the Neurospora crassa plasma membrane has been reconstituted by a gel filtration method into lipidic vesicles using sodium deoxycholate as the detergent. Reconstitution was performed for lipid/ATPase ratios ranging from 1000:1 to 5:1 (ww). Whatever the lipid/ATPase ratio, the ATPase molecules completely associate with the lipid vesicles. The ATPase specific activity is identical for all proteoliposomes regardless of the lipid/ATPase ratio, but the H+ transport decreases at high protein/lipid ratios, suggesting that the proteoliposomes are more leaky to H+ as the amount of protein inserted into the lipidic membrane increases. Analysis of the fragments generated by trypsin proteolysis in the presence and in the absence of MgATP + vanadate indicate that most of the reconstituted ATPase molecules are able to assume the transition state of the enzyme dephosphorylation reaction, and are therefore functional. The orientation (inside-out or rightside-out) of the ATPase molecules in the vesicles is independent of the lipid/ATPase ratio chosen for the reconstitution. For all the lipid/ATPase ratios tested, most of the ATPase molecules (> 99%) expose their cytoplasmic side to the outside of the n.-constituted proteoliposomes. The size of the vesicles increases parallel to the ATPase amount. Although the H+ leakiness of our preparation at low lipid/protein ratios prevents proton pumping measurements, the reconstitution procedure described here has the main advantage on other procedures to allow the obtention of vesicles at high protein-to-lipid ratios, facilitating further structural characterization of the ATPase by biochemical and biophysical techniques. Therefore, the procedure described here could be of general interest in the field of membrane protein study

Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

<p>Abstract</p> <p>Background</p> <p>Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool.</p> <p>Methods</p> <p>We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3).</p> <p>Results</p> <p>Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel.</p> <p>Conclusions</p> <p>Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.</p

Contribution à l'étude de la structure moléculaire de l'ATPase à protons de la membrane plasmique de Neurospora crassa

Doctorat en sciences médicalesinfo:eu-repo/semantics/nonPublishe

Fourier transform infrared spectroscopy study of the secondary structure of the reconstituted Neurospora crassa plasma membrane H(+)-ATPase and of its membrane-associated proteolytic peptides.

We reconstituted purified plasma membrane H(+)-ATPase from Neurospora crassa into soybean phospholipid vesicles (lipid/ATPase ratio of 5:1 w/w). The proteoliposomes contained an active ATPase, oriented inside-out. They were subjected to proteolysis by using Pronase, proteinase K, trypsin, and carboxypeptidase Y. Fourier transform infrared attenuated total reflection spectroscopy indicates that the amount of protein remaining after hydrolysis and elimination of the extramembrane domain of ATPase represents about 43% of the intact protein. The secondary structure of intact ATPase and of the membrane-associated domain of ATPase was determined by infrared spectroscopy. The membrane domain shows a typical alpha-helix and beta-sheet absorption. Polarized infrared spectroscopy reveals that the orientation of the helices is about perpendicular to the membrane. Amide hydrogen/deuterium exchange kinetics performed for the intact H(+)-ATPase and for the membrane-associated domain demonstrate that this part of ATPase shows less accessibility to the solvent than the entire protein but remains much more accessible to the solvent than bacteriorhodopsin membrane segments.Journal Articleinfo:eu-repo/semantics/publishe

Tertiary conformational changes of the Neurospora crassa plasma membrane H(+)-ATPase monitored by hydrogen/deuterium exchange kinetics. A Fourier transformed infrared spectroscopy approach.

Attenuated total reflection Fourier transform infrared spectroscopy of hydrated films of the Neurospora crassa plasma membrane H(+)-ATPase has been used to monitor the alpha-helix and beta-sheet contents and amide hydrogen exchange rates of the enzyme in the absence of ligands or locked in several stages of the enzyme catalytic cycle by MgADP, Mg-vanadate, and MgATP-vanadate. No difference larger than 2% was found in the alpha-helix or beta-sheet content of the H(+)-ATPase in different conformational states. However, when the rate of hydrogen/deuterium exchange monitored by the evolution of the area of amide II and amide II' is decomposed into three components, the number of amide protons characterized by a short exchange rate (1.1 min) falls from 38% of the protein amide protons (or 37% in the presence of Mg2+ alone) to 24-27% in the presence of Mg-vanadate and MgATP-vanadate and to 19% in the presence of MgADP. These results suggest that the conformational changes known to occur when the H(+)-ATPase interacts with the above ligands are predominantly tertiary structure changes.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Fifth DSRG symposium at CHU UCL Namur, 18/10/2019. “Centralization of injectables and robotization”

The physico-chemical stability of an injectable preparation (IV) is conditioned by different parameters. A collaboration between the pharmacy, the chemistry laboratory and the statisticians of the scientific support unit was established in 1996, in order to carry out long-term chemical stability studies of commonly used IVs and to be able to take charge of their preparation in pharmacy. In 24 years of activity, the Drug Stability Research Group (DSRG) tested 39 IV at different concentration and temperature of storage. The DSRG has organized an annual symposium since 2015. The theme of the 2019 edition was devoted to the robotization of injectable reconstitution operations, focused on their impact on the workplace and the existing equipment

La culture des plantes au Moyen Âge. Hildegarde de Bingen, une femme au savoir précurseur

National audienc

Secondary structure of the membrane-bound form of the pore-forming domain of colicin A. An attenuated total-reflection polarized Fourier-transform infrared spectroscopy study.

The structure of the pore-forming domain of the bacterial toxin colicin A was studied by attenuated total-reflection polarized Fourier-transform infrared spectroscopy. This channel-forming fragment interacts with dimyristoylglycerophosphoglycerol (Myr2GroPGro) vesicles and forms disk-like complexes. Analysis of the shape of the amide I' band indicates that its secondary structure is not affected by the pH 5.0-7.2. However, 5-10% of the peptide amino acids adopt an alpha-helical structure upon complex formation with Myr2GroPGro, while the random-coil and beta-sheet structure contents decrease. Interestingly, the increase in alpha-helical content is essentially due to an increase in the high-frequency component of the alpha-helical domain of amide I'. The fact that only this component was 90 degrees polarized (i.e. the helix is parallel to the acyl chain) suggests that only this particular type of helix is associated with the Myr2GroPGro bilayer.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

"La culture des plantes au Moyen Âge. Hildegarde de Bingen, une femme au savoir précurseur"

National audienc

Differences in primary structure among five phospholipases A2 from Heloderma suspectum

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