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 arbejde blev støttet af den canadiske Institute of Health Research. CSC blev finansieret af et postdoc-stipendium fra Natural Sciences and Engineering Research Council of Canada. FD er en Tier II Canada Research Chair.

<html> Samlet Rekonstitueringsprocessen Rekonstitueringen starter ved blanding af membranen scaffold protein (MSP) med det oprensede MalFGK 2-kompleks i nærvær af detergent-solubiliserede phospholipider. Trinnet efterfulgt af langsom fjernelse af detergenten med et adsorberende polystyren materiale kaldet Bio-Beads eller Amberlite (figur 1). Den automatiske montage proces sker sandsynligvis på grund af de apolære interaktioner mellem de hydrofobe phospholipider, d...

<ol>
	<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 procedure til rekonstituering af maltose transportør i nanodiscs. Transportvirksomheden er ATPase aktiv og samspillet med det opløselige bindingspartner MalE kan genskabes (Figur 3). Den vellykkede rekonstituering af transportøren i nanodiscs bane vej for yderligere biofysiske og biokemiske analyser. Af særlig interesse vil være en systematisk analyse af Malk ATPase og maltose transport aktivitet i vaskemiddel, liposom og nanodiscs. ABC-transportører ændrer konformationer un...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Amicon Ultra-4 50K centrifugalfilterenhed </td><td> Millipore </td><td> UFC805008 </td><td> Følg producentens protokol for korrekt anvendelse </td></tr><tr><td> Bio-Beads SM-2 adsorbens </td><td> Bio-Rad </td><td> 152-3920 </td><td></td></tr><tr><td> E. coli samlede lipider </td><td> Avanti Polar Lipids </td><td> 100500C </td><td> Opløst i chloroform, håndtere afhængigt af et organisk opløsningsmiddel </td></tr><tr><td> Ni sepharose HP-harpiks </td><td> GE Healthcare </td><td> 17-5268-01 </td><td></td></tr><tr><td> Fosfor standardopløsning </td><td>Sigma-Aldrich </td><td> P3869 </td><td></td></tr><tr><td> pMSP1D1 </td><td> Addgene </td><td> 20.061 </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> Tabel I. Specifikke reagenser. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Navn </td><td> Sammensætning </td><td> Kommentarer </td></tr><tr><td> DDM lager </td><td> 10% vægt / volumen DDM </td><td> Resuspender i milliQ vand og opbevares ved -20 ° C </td></tr><tr><td> MalFGK 2 lager </td><td> 1-2 mg / ml 50 mM Tris-HCI, pH7.9 100 mM NaCI 10% v / v glycerol 0,03% vægt / volumen DDM </td><td> Opbevares ved -70 ° C efter rensning </td></tr><tr><td> MSP lager </td><td> 10-15 mg / ml 50 mM Tris-HCI, pH7.9 100 mM NaCI 10% v / v glycerol </td><td> Opbevares ved -70 ° C efter oprensning på &lt;1 ml alikvoter og undgå overdrevne fryse / optøningscykler </td></tr><tr><td> Phospholipid lager </td><td> 5 nM E. coli totale lipider 0,5% vægt / volumen (10 mM) DDM 50 mM Tris-HCI, pH 7,9 50 mM NaCI </td><td> Opbevares ved 4 ° C i 1 uge </td></tr><tr><td> TS buffer </td><td> 50 mM Tris-HCI, pH 7,9 50 mM NaCI </td><td> Opbevares ved 4 ° C </td></tr><tr><td> TSG10 buffer </td><td> 50 mM Tris-HCI, pH7.9 100 mM NaCI 10% v / v glycerol </td><td> Opbevares ved 4 ° C </td></tr><tr><td> TSG20 buffer </td><td> 50 mM Tris-HCI, pH 8 100 mM NaCI 20% v / v glycerol </td><td> Opbevares ved 4 ° C </td></tr><tr><td> TSGD buffer </td><td> 50 mM Tris-HCI, pH7.9 100 mM NaCI 10% v / v glycerol 0,03% vægt / volumen DDM </td><td> Opbevares ved 4 ° C, og der tilsættes DDM lige før brug </td></tr><tr><td colspan="3"> Tabel II. Solution opskrifter. </td></tr></tbody></table></td></tr></tbody></table>

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

Det nanodisc er en skiveformede partikel (~ 10-12 nm stor) at fælde membranproteiner i et lille plaster på phospholipid dobbeltlag. The nanodisc er en særlig attraktiv mulighed for at studere membranproteiner, specielt i forbindelse med ligand-receptor-interaktioner. Fremgangsmåden udviklet af Sligar og kolleger er baseret på de amfipatiske egenskaber af et manipuleret højt a-helisk scaffold protein fra apolipoprotein A1. De hydrofobe overflader af stillads-proteinet interagerer med fedtacyl sidekæder af lipiddobbeltlaget henviser polaregnene står det vandige miljø. Analyser af membranproteiner i nanodiscs har betydelige fordele i forhold liposom fordi partiklerne er små, ensartede og vandopløseligt. Endvidere kan biokemiske og biofysiske metoder normalt forbeholdt opløselige proteiner anvendes, og fra begge sider af membranen. I denne visuelle protokol, vi præsenterer en trin-for-trin rekonstituering af et godt kendetegnterized bakteriel ABC transportvirksomheden, mandlige-MalFGK 2-kompleks. Dannelsen af ​​skiven er en selvsamling proces, der afhænger af hydrofobe interaktioner, der finder sted ved den gradvise fjernelse af detergenten. Vi beskriver de væsentlige trin, og vi fremhæver betydningen af ​​at vælge en korrekt protein-til-lipidforhold for at begrænse dannelsen af ​​aggregater og større polydisperse liposom-lignende partikler. Simple kvalitetskontrol, såsom gelfiltreringschromatografi, nativ gelelektroforese og dynamisk lysspredning spektroskopi sikre, at skiverne er blevet korrekt rekonstitueres.

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 skiveformede partikler, der inkorporerer membranproteiner i et lille plaster på phospholipid dobbeltlag. Vi giver en visuel protokol, der viser trin for trin inkorporering af MalFGK2 transportør til en skive.

En trin-for-trin metode til at rekonstituere en ABC Transporter ind i 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 ...

Research

JoVE Journal

Biology

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

En enkel metode til Imaging Arabidopsis Blade Brug Perfluorodecalin som en infiltrativ Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Rekonstituering af et Kv Channel i lipidmembraner for strukturelle og funktionelle studier

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Rekonstituering af et transmembranprotein, Voltage ionkanal, KvAP, i Giant unilamelvesikler til mikroskopi og Patch clamp-forsøg

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Procedure for at vurdere effektiviteten af ​​flokkuleringsmidler til fjernelse af dispergerede partikler fra planteekstrakter

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

En effektiv In Vitro gennemførelse metode af en Transcriptionally regulerede sovende skønhed System pakket ind i en Integration defekte Lentiviral vektor

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

En luft-flydende Interface Bronchial Epitel Model for realistisk, gentagen indånding Eksponering for luftbårne partikler til toksicitet Test

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Tøjrede Bilayer Lipid Membraner til at overvåge varmeoverførsel mellem Gold Nanopartikler og Lipid Membraner

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...