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

Undersøgelserne vedrørende KvAP i Jiang lab har fået betydelig hjælp fra Dr. Roderick MacKinnon laboratorium på Rockefeller University. En særlig tak til Dr. Kathlynn Brown og Michael McQuire for deres råd og hjælp på vores fag-screen eksperimenter. Dette arbejde blev støttet af tilskud fra NIH (GM088745 og GM093271 til Q-XJ) og AHA (12IRG9400019 til Q-XJ).

1.. Ekspression og oprensning af KvAP Channel (figur 1) <ol><li> Forberedelse Work - Dag 0 <ol><li> Skyl glasflasker for bakteriekulturen med deioniseret vand (DIH 2 O) og MilliQ H 2 O (MQH 2 O) for at fjerne spor af rengøringsmiddel fra almindelig opvask. </li><li> Autoklave 1,000 ml LB-medium i 2,8 L Erlenmeyerkolber (i alt to-liters kultur som et eksempel her). Lav hårdhed af vandet blev fundet at være vigtig for den vellykkede dyrkning af de transformerede bakterier. </li><li> A...

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Rekonstituering af KvAP kanaler i forskellige membraner har været anvendt i flere undersøgelser 8-10. Efter ideen om at sikre fordelingen af ​​lipider mellem vaske / lipid blandede miceller og protein / detergent / lipid blandede miceller, er vi i stand til at nå op på næsten fuldstændig opløst KvAP i membraner lavet af meget forskellige lipider. Hver tetramere KvAP kanal behov ~ 100 lipid molekyler til fuldt ud at dække sit transmembrane domæne. Det væsentlige krav er at tillade nok lipidmoleky...

Celler udveksle materialer og information med deres omgivelser gennem funktionerne af specifikke membranproteiner 1.. Membranproteiner i cellemembraner fungerer som pumper, kanaler, receptorer, intramembrane enzymer, linkere og strukturelle støtter tværs membraner. Mutationer, der påvirker membranproteiner har været relateret til mange humane sygdomme. Faktisk har mange membranproteiner været de primære lægemiddelkandidater, fordi de er vigtige og let tilgængelige i cellemembraner. Det er derfor meget vigtigt at forstå strukturen og funktionen af ​​forskellige membranproteiner i membraner, og gøre det muligt at udforme nye metoder til at afhjælpe de skadelige virkninger fra de mutante proteiner i humane sygdomme. Lipider omgiver alle membranproteiner integreret i dobbeltlag 2, 3. I eukaryote membraner, er de forskellige typer af lipider kendt for at være organiseret i mikrodomæner 4, 5.Mange membranproteiner viste sig at være fordelt blandt disse mikrodomæner samt voluminøse flydende fase af membraner 3, 6. Mekanismen bag organiseringen af ​​mikrodomæner og levering af membranproteiner ind i dem, og den fysiologiske betydning af sådanne distributioner er helt klart vigtigt, men forbliver dårligt forstået. En større tekniske vanskeligheder i at studere lipid effekter på membranproteiner er pålidelig rekonstituering af biokemisk oprensede membranproteiner i membraner af velkontrollerede lipid sammensætning, således at næsten alle rekonstitueres proteiner er funktionelle 7.. I de sidste par år har vi udviklet metoder til at rekonstruere prototype spændingsstyret kaliumkanal fra A. pernix (KvAP) i forskellige membran systemer til strukturelle og funktionelle studier 8-10. Dataene fra andre og os sammen viste, at lipiderne er sandsynligvis en bestemmende faktor i konformationelle ændringer i spænding-sensingdomæner af en spænding ionkanal og kan forme strukturerne af nogle af disse kanaler 11. I den næste, vil vi give en detaljeret beskrivelse af vores metoder og vil tilbyde kritiske tekniske tips, der vil sandsynligvis sikre en vellykket reproduktion af vores resultater samt en udvidelse af vores metoder til studier af andre membranproteiner.

<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>
 </tr>
 <tr>
 <td>Aprotinin</td>
 <td>RPI Corp.</td>
 <td>A20550-0.05</td>
 <td></td>
 </tr>
 <tr>
 <td>Leupeptin</td>
 <td>RPI Corp.</td>
 <td>L22035-0.025</td>
 <td></td>
 </tr>
 <tr>
 <td>Pepstatin A</td>
 <td>RPI Corp.</td>
 <td>P30100-0.025</td>
 <td></td>
 </tr>
 <tr>
 <td>PMSF</td>
 <td>SIGMA</td>
 <td>P7626</td>
 <td></td>
 </tr>
 <tr>
 <td>Dnase I</td>
 <td>Roche</td>
 <td>13407000</td>
 <td></td>
 </tr>
 <tr>
 <td>Bio-Bead SM-2</td>
 <td>Bio-Rad</td>
 <td>152-3920</td>
 <td></td>
 </tr>
 <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

At undersøge lipid-protein-interaktion i en reduktionistisk måde, er det nødvendigt at inkorporere membranproteiner i membraner af veldefineret lipidsammensætning. Vi studerer lipid-afhængige gating effekter i en prototype spændingsstyret kalium (Kv) kanal og har arbejdet detaljerede procedurer at rekonstruere kanalerne i forskellige membran-systemer. Vores rekonstituering procedurer tager hensyn til både vaskemiddel-induceret fusion af vesikler og sammensmeltningen af ​​protein / detergent miceller med lipid / detergent blandede miceller samt betydningen af ​​at nå en ligevægt fordeling af lipider blandt protein / detergent / lipid og detergent / lipid blandede miceller. Vores data antydede, at indsættelsen af ​​kanalerne i lipidvesiklerne er forholdsvis tilfældigt i orienteringer, og rekonstituering effektivitet er så høj, at ingen påviselige protein aggregater blev set i fraktionering eksperimenter. Vi har udnyttet rekonstituered kanaler til at bestemme de konformationelle tilstande af kanalerne i forskellige lipider, registrerer elektriske aktiviteter af et lille antal kanaler er indarbejdet i plane lipiddobbeltlag, skærm til kropsbygning-specifikke ligander fra en fag-viste peptid bibliotek og støtte væksten i 2D krystaller af kanalerne i membranerne. Rekonstitueringen her beskrevne procedurer kan tilpasses til at studere andre membranproteiner i lipiddobbeltlag, især til undersøgelse af lipid effekter på eukaryote spændingsstyrede ionkanaler.

Den generelle strøm af forsøgene til rensning af KvAP kanal til biokemisk homogenitet er beskrevet i figur 1A. Typiske prøver under ekspression og oprensning af proteinet er vist i SDS-PAGE-gel i figur 1B. Proteinet efter IMAC oprensning er forholdsvis ren. Udbyttet af KvAP kanal er omkring 1,0 mg / liter kultur. Opløsning af lipidvesikler med rengøringsmidler skal arbejdes ud for hvert par af lipid vs vaskemiddel. Solubilisering af små unilamellare ve...

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

Procedurer for rekonstituering af en prototype spændingsstyret kaliumkanal i lipidmembraner beskrevet. De rekonstituerede kanaler er egnet til biokemiske analyser, elektriske optagelser, ligand screening og elektron krystallografiske studier. Disse metoder kan have generelle anvendelser til de strukturelle og funktionelle studier af andre membranproteiner.

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 ...

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Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

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

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 ...

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

Anvendelse af en robot til Højkapacitetsforskning Krystallisation af membranproteiner i Lipidiske mesofaser

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

Høst og Cryo-køling Krystaller af membranproteiner dyrket i Lipidiske mesofaser for Structure Bestemmelse ved makromolekylær krystallografi

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

Isolering og Kv-optagelser i murine atrielle og ventrikulære cardiomyocytter

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 Produktion og oprensning af rekombinant murin Kindlin-3 fra Insect Celler til Biofysiske Studies

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

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 ...