Name: reconstitution of a kv channel into lipid membranes for structural and functional studies
Uploaded: 2013-07-13T13:00:00
Description: Watch this Scientific Journal Video about Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies at JoVE.com

Studiene på KvAP i Jiang lab har fått betydelig hjelp fra dr. Roderick MacKinnon laboratorium ved Rockefeller University. Spesiell takk går til Dr. Kathlynn Brown og Michael McQuire for sine råd og hjelp på våre fag-skjermen eksperimenter. Dette arbeidet ble støttet med tilskudd fra NIH (GM088745 og GM093271 til Q-XJ) og AHA (12IRG9400019 til Q-XJ).

En. Uttrykk og rensing av KvAP Channel (Figur 1) <ol><li> Forberedelse Work - Dag 0 <ol><li> Skyll glass kolber for bakteriekultur med avionisert vann (Dih 2 O) og MilliQ H 2 O (MQH 2 O) for å fjerne spor av vaskemiddel fra generell oppvask. </li><li> Autoklav 1000 ml LB-medium i 2,8 l Erlenmeyer-kolber (totalt to-liters kultur som et eksempel her). Lav hardhet av vannet ble funnet å være viktig for en vellykket kultur av de transformerte bakterier. </li><li> Autoklav 100 ml LB medi...

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Tilberedning av KvAP kanaler i forskjellige membraner har vært brukt i flere studier 8-10. Etter ideen med å sikre fordelingen av lipider mellom vaskemiddel / lipid blandede miceller og proteinet / vaskemiddel / lipid blandet miceller, er vi i stand til å nå nesten fullstendig rekonstituering av KvAP inn membraner laget av meget forskjellige lipider. Hver tetramerisk KvAP kanalene må ~ 100 lipidmolekyler å fullt ut dekke sin transmembrane domene. Det grunnleggende kravet er å tillate nok lipidmolekyler...

Celler utveksle materiale og informasjon med sine omgivelser gjennom funksjonene til spesifikke membran proteiner en. Membran proteiner i cellemembraner fungere som pumper, kanaler, reseptorer, intramembrane enzymer, linkers og strukturelle støttespillere over membraner. Mutasjoner som påvirker membran proteiner har vært knyttet til mange menneskelige sykdommer. Faktisk har mange membranproteiner vært de primære stoff mål fordi de er viktige og lett tilgjengelig i cellemembraner. Det er derfor svært viktig å forstå strukturen og funksjonen til ulike membran proteiner i membraner, og gjøre det mulig å utvikle nye metoder for å lindre skadevirkningene fra den muterte proteiner i menneskelige sykdommer. Lipider surround alle membran proteiner integrert i to bilayers, tre. I eukaryote membraner, vises de forskjellige ulike typer av lipider kjent for å være organisert i mikroområder 4, 5.Mange membranproteiner ble vist å bli distribuert blant disse mikroområder samt voluminøse fluidfase av membranene 3, 6. Mekanismen bak organiseringen av mikroområder og levering av membran proteiner inn i dem og den fysiologiske betydningen av slike utdelinger er helt klart viktig, men er fortsatt dårlig forstått. En stor tekniske problemer i å studere lipid effekter på membran proteiner er pålitelig rekonstituering av biokjemisk renset membran proteiner i membraner av godt kontrollert lipid sammensetning, slik at nesten alle rekonstituert proteiner er funksjonelle 7. I de siste årene, har vi utviklet metoder for å rekonstruere prototypen spenning gated kalium kanal fra A. pernix (KvAP) i ulike membransystemer for strukturelle og funksjonelle studier 8-10. Dataene fra andre og oss sammen viste at lipidene er sannsynligvis en determinant i konformasjonsendringer i spennings-sensingdomener av et spenningsstyrte ionekanal og kan forme strukturer av noen av disse kanalene 11. I den neste, vil vi gi en detaljert beskrivelse av våre metoder og vil tilby kritiske tekniske tips som trolig vil sikre en vellykket reproduksjon av våre resultater, samt utvidelse av våre metoder til studier av andre membran proteiner.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tryptone</td>
 <td>RPI Corp.</td>
 <td>T60060</td>
 <td></td>
 </tr>
 <tr>
 <td>Yeast Extract</td>
 <td>RPI Corp.</td>
 <td>Y20020</td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Fisher</td>
 <td>S271-3</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>RPI Corp.</td>
 <td>T60040</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium Chloride</td>
 <td>Fisher</td>
 <td>BP366-500</td>
 <td></td>
 </tr>
 <tr>
 <td>n-Dodecyl-&beta;-D-Maltoside</td>
 <td>Affymetrix</td>
 <td>D322S</td>
 <td>Sol-grade</td>
 </tr>
 <tr>
 <td>n-Octyl-&beta;-D-Glucoside </td>
 <td>Affymetrix</td>
 <td>O311</td>
 <td>Ana-grade</td>
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 <tr>
 <td>Aprotinin</td>
 <td>RPI Corp.</td>
 <td>A20550-0.05</td>
 <td></td>
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 <td>Leupeptin</td>
 <td>RPI Corp.</td>
 <td>L22035-0.025</td>
 <td></td>
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 <tr>
 <td>Pepstatin A</td>
 <td>RPI Corp.</td>
 <td>P30100-0.025</td>
 <td></td>
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 <tr>
 <td>PMSF</td>
 <td>SIGMA</td>
 <td>P7626</td>
 <td></td>
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 <tr>
 <td>Dnase I</td>
 <td>Roche</td>
 <td>13407000</td>
 <td></td>
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 <tr>
 <td>Bio-Bead SM-2</td>
 <td>Bio-Rad</td>
 <td>152-3920</td>
 <td></td>
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 <tr>
 <td>HEPES</td>
 <td>RPI Corp.</td>
 <td>H75030</td>
 <td></td>
 </tr>
 <tr>
 <td>POPE</td>
 <td>Avanti Polar Lipids</td>
 <td>850757C</td>
 <td></td>
 </tr>
 <tr>
 <td>POPG</td>
 <td>Avanti Polar Lipids</td>
 <td>840457C</td>
 <td></td>
 </tr>
 <tr>
 <td>DOGS</td>
 <td>Avanti Polar Lipids</td>
 <td>870314C</td>
 <td></td>
 </tr>
 <tr>
 <td>DMPC </td>
 <td>Avanti Polar Lipids</td>
 <td>850345C</td>
 <td></td>
 </tr>
 <tr>
 <td>Biotin-DOPE </td>
 <td>Avanti Polar Lipids</td>
 <td>870282C</td>
 <td></td>
 </tr>
 <tr>
 <td>DOTAP</td>
 <td>Avanti Polar Lipids</td>
 <td>890890C</td>
 <td></td>
 </tr>
 <tr>
 <td>NeutrAvidin agarose beads </td>
 <td>Piercenet</td>
 <td>29200</td>
 <td></td>
 </tr>
 <tr>
 <td>Dialysis Tubing</td>
 <td>Spectrum Laboratories, Inc</td>
 <td>132-570</td>
 <td></td>
 </tr>
 <tr>
 <td>Pentane</td>
 <td>Fisher</td>
 <td>R399-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Decane</td>
 <td>TCI America</td>
 <td>D0011</td>
 <td></td>
 </tr>
 <tr>
 <td>MTS-PEG5000</td>
 <td>Toronto Research Cemicals</td>
 <td>M266501</td>
 <td></td>
 </tr>
 </tbody>
</table>

reconstitution of a kv channel into lipid membranes for structural and functional studies

For å studere lipid og protein i en reductionistic måte, er det nødvendig å innlemme membran proteiner inn i membraner av veldefinerte lipidsammensetning. Vi studerer lipid-avhengige gating effekter i en prototype spenning-gated potassium (Kv) kanal, og har jobbet ut detaljerte prosedyrer for å gjeninnføre kanalene i ulike membransystemer. Våre reconstitution prosedyrer tar hensyn til både vaskemiddel-indusert fusjon av blemmer og sammensmeltingen av protein / vaskemiddel micelles med lipid / vaskemiddel blandet micelles samt betydningen av å nå en likevekt fordeling av lipider mellom protein / vaskemiddel / lipid og vaskemiddel / lipid blandet micelles. Våre data antyder at innsetting av kanalene i de lipidvesikler er forholdsvis slumpmessig i orienteringer, og rekonstituering virkningsgrad er så høy at ingen påviselige protein aggregater ble observert i eksperimenter fraksjonering. Vi har benyttet rekonstituererd kanaler for å bestemme de konformasjonsforandringer statene kanalene i ulike lipider, registrere elektriske aktiviteter av et lite antall kanaler som inngår i planar lipid bilayers, skjerm for eksteriør-spesifikke ligander fra en fag-displayed peptid bibliotek og støtte veksten av 2D krystaller av kanalene i membranene. Tilberedning beskrevet her kan tilpasses for å studere andre membran proteiner i lipid bilayers, spesielt for undersøkelse av lipid effekter på eukaryote spenningsstyrte ionekanaler.

Den generelle strømmen av forsøkene for å rense KvAP kanal inn i biokjemisk homogenitet er beskrevet i figur 1A. Typiske eksempler under ekspresjon og rensing av proteinet er vist i SDS-PAGE-gel i figur 1B. Proteinet etter IMAC rensing er relativt ren. Utbyttet av KvAP kanalen er omtrent 1,0 mg / liter kultur. Oppløsning av lipidvesiklene med vaskemidler må utarbeides for hvert par av lipid vs oppvaskmiddel. Av oppløsningen av små unilamillære vesikl...

Watch this Scientific Journal Video about Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies at JoVE.com

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

Prosedyrer for fullstendig rekonstituering av en prototyp spenningsstyrte kaliumkanaler inn i lipidmembraner, er beskrevet. De rekonstituerte kanaler er egnet for biokjemiske analyser, elektriske innspillinger, ligand screening og elektron krystallografisk studier. Disse metodene kan ha generell anvendelse til de strukturelle og funksjonelle studier av andre membranproteiner.

Tilberedning av en Kv Channel i lipid membraner for strukturelle og funksjonelle studier

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Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

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

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 ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Bruk av en robot for High-throughput Krystallisering av membran proteiner i Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

Høsting og Cryo-kjøling Krystaller av membran proteiner Grown i Lipidic Mesophases for strukturbestemmelse av Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Isolasjon og Kv Channel Recordings i Murine Atrial og ventrikulær cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Effektiv produksjon og rensing av rekombinant mus Kindlin-3 fra insektceller for biofysiske studier

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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

Tilberedning av et trans Protein, den Spenning ionekanal, KvAP, inn Giant unilamellær Blemmer for Mikros og Patch Clamp Studier

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