51 research outputs found
Structural and functional characterization of PorA and PorH : the two major porins from Corynebacterium glutamicum
No full textPorA (5 kDa) et PorH (7kDa) sont les deux protéines membranaires majeures de la membrane externe de Corynebacterium glutamicum qui appartient au groupe supragénérique des bactéries Gram-positives contenant plusieurs agents pathogènes i.e. Mycobacterium tuberculosis, M. leprae et C. diphtheriae. Les deux protéines forment des canaux ioniques hétéromériques et présentent la particularité d'avoir une modification post-traductionnelle : estérification par l'acide mycolique. Les deux protéines ont été produites dans deux systèmes d'expression : chez C. glutamicum et dans un système acellulaire utilisant des extraits de E. coli. La présence ou l'absence de modification post-traductionnelle, sur les protéines produites in vivo et in vitro a été caractérisée par spectrométrie de masse MALDI-TOF. Les spectres de dichroïsme cellulaire et de RMN de PorA et PorH uniformément marquées et solubilisées en micelles de LDAO sont caractéristiques de protéines mono-disperses, partiellement structurées, et de qualité compatible avec une détermination de structure par RMN. Le test fonctionnel des protéines, par mesure de conductivité ionique après reconstitution dans une membrane lipidique (technique dite de BLM pour " black lipid membrane ") a montré que a) la modification post-traductionnelle de PorA par un acide mycolique est essentielle (contrairement à celle de PorH) b) la présence simultanée de PorA et de PorH est requise pour la formation d'un canal ionique voltage dépendant typique d'une porine. Afin de mieux comprendre l'importance de l'acide mycolique pour l'activité canal ionique, le complexe protéique PorA-PorH a été reconstitué dans son environnement naturel. Les principaux lipides de la membrane externe du C. glutamicum [Tréhalose dimycolate (TDM), tréhalose monomycolate (TMM) et cardiolipide (CL)] ont été extraits et purifiés par chromatographie d'adsorption et échange d'ions, sous forme protonée et sous forme perdeutériée. Après formation de protéoliposomes les propriétés membranaires de TDM seul ou en mélange avec CL ont été étudiées RMN du deutérium, diffusion dynamique de la lumière et microscopie électronique. L'insertion de PorA et PorH dans des vésicules de TDM a permis de mettre en évidence la reconstitution de l'hétéro-oligomère (contrairement aux micelles de LDAO). Ceci ouvre la voie à la détermination de structure 3D du complexe PorA-PorH fonctionnel par RMN solide et/ou liquide.PorA (5 kDa) and PorH (7 kDa) are two major membrane proteins from the outer membrane of Corynebacterium glutamicum which belongs to the suprageneric group of Gram-positive bacteria containing number of human pathogens such as Mycobacterium tuberculosis, M. leprae and C. diphtheriae. Both PorA and PorH have been shown to form heteromeric ion channels and to be post-translationally modified by mycolic acids (a-alkyl, beta-hydroxy fatty acids).
Both proteins were produced in their natural host with mycolic acid modification, as well as in E. coli based continuous exchange cell-free expression system and thus devoid of mycolic acid modification. The presence or absence of mycolic acid modification on in vivo and in vitro expressed proteins was confirmed by MALDI-TOF mass spectrometry. CD and NMR spectra of 15N/13C uniformly labeled PorA and PorH solubilized in LDAO micelles indicated mono-dispersed and partially folded proteins, compatible with structure determination by NMR. However, functional assays (via black lipid membrane ion-channel conductance measurements) confirmed that a complex associating both proteins is required for function and that the mycolic acid modification on PorA (but not PorH), is an absolute requirement for the formation of a voltage dependent ion-channel. To understand further the importance of covalent or non-covalent interaction of their natural lipid environment on the complex formation, the major C. glutamicum outer membrane lipids [Trehalose dimycolate (TDM), Trehalose monomycolate (TMM) and Cardiolipin (CL)] were purified using adsorption and ion exchange chromatography, both in protonated and perdeuterated form. Prior to proteoliposome reconstitution, the membrane forming properties of TDM alone or in mixture with CL were studied by 2H-NMR, Dynamic Light Scattering and Electron Microscopy. Furthermore, after in vitro reconstitution of PorA and PorH in TDM vesicles (and not in LDAO micelles or DMPC vesicles), evidence for the formation of the hetero oligomeric complex was observed. The 3D structure determination, by liquid and/or solid state NMR, of a functional PorA-PorH complex in its natural lipid environment is now feasible
The Role of the Sensing Domain (S1-S4) in TRPM8 Temperature and Menthol Dependent Gating
No full textImplications of Human TRPM8 Channel Gating from Sensing Domain and Menthol Binding Studies
No full textCaractérisation structurale et fonctionnelle de PorA et PorH (les deux majeures porines de corynebacterium glutamicum)
No full textTOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF
Enhancement Effects of Biosurfactant Produced by Pseudomonas aeruginosa MTCC 2297 and Pseudomonas fluorescens on Sugar Cane Bagasse Composting
No full textBiosurfactant produced by Pseudomonas aeruginosa MTCC2297 and Pseudomonas fluorescens (a Mangrove forest isolate) was added to the Sugar cane bagasse decomposing process to initiate and enhance the production of reducing sugars. Both isolates produced a glycolipid biosurfactant, namely a rhamnolipid that was confirmed by biochemical and analytical studies. The surface tension of fermentation broth reduced from 73 to 34.2 mN/m by P. aeruginosa MTCC 2297 and from 76 to 29 mN/m by P. fluorescens. Sugar cane bagasse decomposing process was attempted by two different methods, adding pure rhamnolipid and on-site production of it were compared. The study revealed that rhamnolipid concentration of 0.75gl-1 was optimum for composting process and the optimum temperature for compost pile production was 32°Cfor the first 48h subsequently 35°C for the next 48h. At the optimum temperature for this two-stage fermentation, the production of reducing sugar could be increased to 15.73 gl-1 for P. aeruginosa MTCC 2297 and for the P. fluorescens reducing sugar increased to 15.88 gl-1, both experiments resulted in higher values than that of the treatment without rhamnolipid sample. The results indicate that application biosurfactant could possibly reduce the composting period. As the compost pile production of rhamnolipid does not require the purification process, the production cost reduced significantly.--------------------------------------------------------------------------------------------------------Department of Microbiology, Faculty of Agriculture, Annamalai University, Annamalai Nagar, Chidambaram, Tamil Nadu, India-608002. *Corresponding author, Email: [email protected], Mob: +91-9842059688               Cite This Article As: Parthasarathi, R. and  P. K. Sivakumaar. 2010. Enhancement Effects of Biosurfactant Produced by Pseudomonas aeruginosa MTCC 2297 and Pseudomonas fluorescens on Sugar Cane Bagasse Composting. J. Ecobiotechnol. 2(3): 33-39
Two-state folding of the outer membrane protein X into a lipid bilayer membrane
No full textFolding and insertion of β-barrel membrane proteins into native membranes is efficiently catalyzed by β-barrel assembly machineries. Understanding this catalysis requires a detailed description of the corresponding uncatalyzed folding mechanisms, which however have so far remained largely unclear. Here, we resolve folding and membrane insertion of the E. coli outer membrane protein X (OmpX) into 1,2-didecanoyl-sn-glycero-3-phosphocholine (PC10:0) membranes at the atomic level. By combining four different experimental techniques, we correlate global folding kinetics with global and local hydrogen bond formation kinetics. Under a well-defined reaction condition, these processes follow single-exponential velocity laws, with rate constants identical within experimental error. The data thus establish at atomic resolution that OmpX folds and inserts into the lipid bilayer of PC10:0 liposomes by a two-state mechanism
Designing Supramolecular Gelators: Challenges, Frustrations, and Hopes
No full textThis article is a personal account of the author, who serendipitously entered the field of supramolecular gels nearly two decades ago. A supramolecular synthon approach in the context of crystal engineering was utilized to develop a working hypothesis to design supramolecular gelators derived from simple organic salts. The activity not only provided a way to occasionally predict gelation, but also afforded clear understanding of the structural landscape of such supramolecular materials. Without waiting for an ab initio approach for designing a gel, a large number of supramolecular gelators derived from organic salts were designed following the working hypothesis thus developed. Organic salts possess a number of advantages in terms of their ease of synthesis, purification, high yield and stability and, therefore, are suitable for developing materials for various applications. Organic salt-based gel materials for containing oil spills, synthesizing inorganic nanostructures and metal nanoparticles, sensing hazardous gas and dissolved glucose, adsorbing dyes, and facilitating drug delivery in self-delivery fashion have been developed. The journey through the soft world of gelators which was started merely by serendipity turned out to be rewarding, despite the challenges and frustrations in the field
The electrostatic core of the outer membrane protein X from E. coli
No full textElectrostatic side chain contacts can contribute substantial interaction energy terms to the stability of proteins. The impact of electrostatic interactions on the structure and architecture of outer membrane proteins is however not well studied compared to soluble proteins. Here, we report the results of a systematic study of all charged side chains of the E. coli outer membrane protein X (OmpX). The data identify three distinct salt-bridge clusters in the core of OmpX that contribute significantly to protein stability in dodecylphosphocholine detergent micelles. The three clusters form an "electrostatic core" of the membrane protein OmpX, corresponding in its architectural role to the hydrophobic core of soluble proteins. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins
Lessons from the interstitial lung disease-India registry: A proposed practical scheme of classification of diffuse parenchymal lung diseases in the Indian subcontinent
No full textThe revelation of interstitial lung disease India registry leads one to consider a revisit into the classification of diffuse parenchymal lung disease that would be clinically relevant for the Indian subcontinent. The author ponders that a simple clinical classification based on steroid sensitivity may be worthwhile
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