Name: a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles
Uploaded: 2012-08-31T12:00:00
Description: Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

Dette arbeidet ble støttet av den kanadiske Institute of Health Research. CSC ble finansiert av en postdoktorstilling fra Natural Sciences and Engineering Research Council of Canada. FD er en Tier II Canada Research Chair.

<html> Samlet Rekonstituering Process Tilberedning Prosessen starter ved å blande membranen scaffoldprotein (MSP) med rensede MalFGK 2 kompleks i nærvær av vaskemiddel-solubilisert fosfolipider. Trinnet er etterfulgt av langsom fjerning av vaskemiddel ved en adsorbent polystyren materiale kalt Bio-perler eller Amberlite (figur 1). Den automatiske forsamlingen prosessen skjer mest sannsynlig på grunn av apolare interaksjoner mellom de hydrofobe fosfolipider, den Ma...

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	<li>Denisov, I. G., Ginkova, Y. V., Lazarides, A. A., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+self-assembly+of+monodisperse+phospholipid+bilayer+Nanodiscs+with+controlled+size.">Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size.</a> J. Am. Chem. Soc. 126, 3477-3487 (2004).</li><li>Boldog, T., Grimme, S., Li, M., Sligar, S. G., Hazelbauer, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+separate+chemoreceptor+oligomeric+states+and+reveal+their+signaling+properties.">Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties.</a> Proc. Natl. Acad. Sci. USA. 103, 11509-11514 (2006).</li><li>Bass, B. J., Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homotropic+cooperativity+of+monomeric+cytochrome+P450+3A4+in+a+nanoscale+native+bilayer+environment.">Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment.</a> J. Biol. Chem. 282, 7066-7076 (2007).</li><li>Alami, M., Dalal, K., Lelj-Garolla, B., Sligar, S. G., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+unravel+the+interaction+between+the+SecYEG+channel+and+its+cytosolic+partner+SecA.">Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA.</a> EMBO J. 26, 1995-2004 (2007).</li><li>Mi, L. -. Z., Grey, M. J., Nishida, N., Walz, T., Lu, C., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+stability+of+the+epidermal+growth+factor+receptor+in+detergent+micelles+and+phospholipid+nanodiscs.">Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs.</a> Biochemistry. 47, 10314-10323 (2008).</li><li>Sch&#228;gger, H., Cramer, W. A., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal. Biochem. 217, 220-230 (1994).</li><li>Dalal, K., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+SecY+translocon+in+Nanodiscs.">Reconstitution of the SecY translocon in Nanodiscs.</a> Methods Mol. Biol. 619, 145-156 (2010).</li><li>Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., Candia, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+assay+for+nanomole+amounts+of+inorganic+phosphate.">An improved assay for nanomole amounts of inorganic phosphate.</a> Anal. Biochem. 100, 95-97 (1979).</li><li>Davidson, A. L., Dassa, E., Orelle, C., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function+and+evolution+of+bacterial+ATP-binding+cassette+systems.">Structure, function and evolution of bacterial ATP-binding cassette systems.</a> Microbiol. Mol. Biol. Rev. 72, 317-364 (2008).</li><li>Bordignon, E., Grote, M., Schneider, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maltose+ATP-binding+cassette+transporter+in+the+21st+century-towards+a+structural+dynamic+perspective+on+its+mode+of+action.">The maltose ATP-binding cassette transporter in the 21st century-towards a structural dynamic perspective on its mode of action.</a> Mol. Microbiol. 77, 1354-1366 (2010).</li><li>Alvarez, F. J., Orelle, C., Davidson, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reconstitution+of+an+ABC+transporter+for+use+in+electron+paramagnetic+resonance+spectroscopy.">Functional reconstitution of an ABC transporter for use in electron paramagnetic resonance spectroscopy.</a> J. Am. Chem. Soc. 132, 9513-9515 (2010).</li><li>Ritchie, T. K., Grinkova, Y. V., Bayburt, T. H., Denisov, I. G., Zolnerciks, J. K., Atkins, W. M., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+in+phospholipid+bilayer+Nanodiscs.">Reconstitution of membrane proteins in phospholipid bilayer Nanodiscs.</a> Methods Enzymol. 464, 211-231 (2009).</li><li>Gl&#252;ck, J. M., Koenig, B. W., Willbold, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+allow+the+use+of+integral+membrane+proteins+as+analytes+in+surface+plasmon+resonance+studies.">Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies.</a> Anal. Biochem. 408, 46-52 (2011).</li><li>Wan, C. -. P. L., Chiu, M. H., Wu, X., Lee, S. K., Prenner, E. J., Weers, P. M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein-induced+conversion+of+phosphatidylcholine+bilayer+vesicles+into+nanodisks.">Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks.</a> Biochim. Biophys. Acta (BBA). 1808, 606-613 (2011).</li><li>Nath, A., Trexler, A. J., Koo, P. K., Miranker, A. D., Atkins, W. M., Rhoades, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+spectroscopy+using+phospholipid+bilayer+Nanodiscs.">Single-molecule fluorescence spectroscopy using phospholipid bilayer Nanodiscs.</a> Methods Enzymol. 472, 89-117 (2010).</li><li>Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochromes+P450+in+Nanodiscs.">Cytochromes P450 in Nanodiscs.</a> Biochim. Biophys. Acta. 1814, 223-229 (2011).</li><li>Zhang, X. X., Chan, C. S., Bao, H., Fang, Y., Foster, L. J., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+and+SILAC-based+mass+spectrometry+to+identify+a+membrane+protein+interactome.">Nanodiscs and SILAC-based mass spectrometry to identify a membrane protein interactome.</a> J. Proteome Res. , (2011).</li></ol>

Vi beskriver en enkel prosedyre for rekonstituering av maltose transporter inn nanodiscs. Transportøren er ATPasen aktiv og samspillet med oppløselig bindingspartner MALE kan gjenskapes (figur 3). Den vellykkede rekonstituering av transportøren i nanodiscs åpne veien for ytterligere biofysiske og biokjemiske analyser. Av særlig interesse vil være systematisk analyse av Malk ATPase og maltose transport aktivitet i vaskemiddel, liposome og nanodiscs. ABC transportører endrer konformasjonen under tr...

<table border="1"><tbody><tr><td> Navnet på reagensen </td><td> Selskapet </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Amicon Ultra-4 50K sentrifugalfilter </td><td> Millipore </td><td> UFC805008 </td><td> Følg produsentens protokoll for riktig bruk </td></tr><tr><td> Bio-perler SM-2 Adsorbent </td><td> Bio-Rad </td><td> 152-3920 </td><td></td></tr><tr><td> E. coli totalt lipider </td><td> Avanti Polar Lipids </td><td> 100500C </td><td> Oppløst i kloroform, håndtak som passer for et organisk oppløsningsmiddel </td></tr><tr><td> Ni sefarose HP harpiks </td><td> GE Healthcare </td><td> 17-5268-01 </td><td></td></tr><tr><td> Fosfor standardløsning </td><td>Sigma-Aldrich </td><td> P3869 </td><td></td></tr><tr><td> pMSP1D1 </td><td> Addgene </td><td> 20061 </td><td></td></tr><tr><td> Superdex 200 HR 10/300 </td><td> GE Healthcare </td><td> 17-5172-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabell I. Spesifikke reagenser. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Navn </td><td> Sammensetning </td><td> Kommentarer </td></tr><tr><td> DDM lager </td><td> 10% w / v DDM </td><td> Resuspender i milliQ vann og oppbevar ved -20 ° C </td></tr><tr><td> MalFGK 2 på lager </td><td> 1-2 mg / ml 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v glyserol 0,03% w / v DDM </td><td> Oppbevar ved -70 ° C etter rensing </td></tr><tr><td> MSP lager </td><td> 10-15 mg / ml 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v glyserol </td><td> Oppbevar ved -70 ° C etter rensing i &lt;1 ml alikvoter og unngå overdreven fryse / tine sykluser </td></tr><tr><td> Fosfolipid lager </td><td> 5 nM E. coli totalt lipider 0,5% w / v (10 mM) DDM 50 mM Tris-HCl, pH 7,9 50 mM NaCl </td><td> Oppbevar ved 4 ° C i 1 uke </td></tr><tr><td> TS buffer </td><td> 50 mM Tris-HCl, pH 7,9 50 mM NaCl </td><td> Oppbevar ved 4 ° C </td></tr><tr><td> TSG10 buffer </td><td> 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v glyserol </td><td> Oppbevar ved 4 ° C </td></tr><tr><td> TSG20 buffer </td><td> 50 mM Tris-HCl, PH8 100 mM NaCl 20% v / v glyserol </td><td> Oppbevar ved 4 ° C </td></tr><tr><td> TSGD buffer </td><td> 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v glyserol 0,03% w / v DDM </td><td> Oppbevar ved 4 ° C og legg DDM like før bruk </td></tr><tr><td colspan="3"> Tabell II. Løsning oppskrifter. </td></tr></tbody></table></td></tr></tbody></table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

Den nanodisc er en skiveformede partikkel (~ 10-12 nm store) som felle membran proteiner i en liten oppdatering av fosfolipid bilayer. Den nanodisc er et spesielt attraktivt alternativ for å studere membran proteiner, spesielt i sammenheng av ligand-reseptor interaksjoner. Metoden utviklet av Sligar og kolleger er basert på amfipatiske egenskapene til en konstruert svært en-helical scaffoldprotein avledet fra apolipoprotein A1. De hydrofobe ansikter av scaffoldprotein samhandle med fettsyreasylgrupper side-kjeder av lipid bilayer mens polarområdene ansikt vandige miljø. Analyser av membran proteiner i nanodiscs har betydelige fordeler fremfor liposom fordi partiklene er små, homogen og vannoppløselig. I tillegg kan biokjemiske og biofysiske metoder normalt reservert til løselige proteiner bli brukt, og fra begge sider av membranen. I denne visuelle protokollen, presenterer vi en steg-for-steg omdanning av et godt karakteriserte d bakteriell ABC transportør, den mannlige-MalFGK 2 kompleks. Dannelsen av platen er en selv-oppsettingen som avhenger hydrofobe interaksjoner finner sted under den progressive fjerning av vaskemiddel. Vi beskriver de grunnleggende trinnene og vi markere betydningen av å velge en riktig protein-til-lipid forholdet for å begrense dannelsen av aggregater og større polydisperse liposom-lignende partikler. Enkelt kvalitetskontroller eksempel gelfiltrering kromatografi, innfødt gelelektroforese og dynamisk lysspredning spektroskopi sikre at platene har blitt riktig rekonstituert.

Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Nanodiscs er små discoid partikler som inneholder membran proteiner i en liten oppdatering av fosfolipid bilayer. Vi tilbyr en visuell protokoll som viser trinn-for-trinn innlemmelse av MalFGK2 transporter inn en plate.

A Step-by-step Metode for Rekonstituering av en ABC Transporter inn Nanodisc lipidpartikler

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

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