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

This work was supported by the Canadian Institute of Health Research. CSC was funded by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. FD is a Tier II Canada Research Chair.

Overall Reconstitution Process
 The reconstitution process starts by mixing the membrane scaffold protein (MSP) with the purified MalFGK2 complex in the presence of detergent-solubilized phospholipids. The step is followed by the slow removal of the detergent by an adsorbent polystyrene material called Bio-Beads or Amberlite (Figure 1). The auto-assembly process occurs most likely because of the apolar interactions between the hydrophobic phospholipids, the MalF...

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We describe a simple procedure for the reconstitution of the maltose transporter into nanodiscs. The transporter is ATPase active and the interaction with the soluble binding partner MalE can be recreated (Figure 3). The successful reconstitution of the transporter into nanodiscs open the way for additional biophysical and biochemical analysis. Of particular interest will be the systematic analysis the MalK ATPase and maltose transport activity in detergent, liposome and nanodiscs. ABC transporters c...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Amicon Ultra-4 50K centrifugal filter</td>
 <td>Millipore</td>
 <td>UFC805008</td>
 <td>Follow manufacturer's protocol for proper use </td>
 </tr>
 <tr>
 <td>Bio-Beads SM-2 Adsorbent</td>
 <td>Bio-Rad</td>
 <td>152-3920</td>
 <td></td>
 </tr>
 <tr>
 <td>E. coli total lipids</td>
 <td>Avanti Polar Lipids</td>
 <td>100500C</td>
 <td>Dissolved in chloroform, handle as appropriate for an organic solvent</td>
 </tr>
 <tr>
 <td>Ni sepharose HP resin</td>
 <td>GE Healthcare</td>
 <td>17-5268-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphorous standard solution</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>Table I. Specific reagents.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
 <table border="1">
 <tbody>
 <tr>
 <td>Name </td>
 <td>Composition</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>DDM stock</td>
 <td>10% w/v DDM</td>
 <td>Resuspend in milliQ water and store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>MalFGK2 stock</td>
 <td>1-2 mg/ml 
 50 mM Tris-HCl, pH7.9 
 100 mM NaCl 
 10% v/v glycerol 
 0.03% w/v DDM</td>
 <td>Store at -70 &deg;C after purification</td>
 </tr>
 <tr>
 <td>MSP stock</td>
 <td>10-15 mg/ml 
 50 mM Tris-HCl, pH7.9 
 100 mM NaCl 
 10% v/v glycerol</td>
 <td>Store at -70 &deg;C after purification in &lt;1 ml aliquots and avoid excessive freeze/thaw cycles</td>
 </tr>
 <tr>
 <td>Phospholipid stock</td>
 <td>5 nM E. coli total lipids 
 0.5% w/v (10 mM) DDM 
 50 mM Tris-HCl, pH 7.9 
 50 mM NaCl</td>
 <td>Store at 4 &deg;C for 1 week</td>
 </tr>
 <tr>
 <td>TS buffer</td>
 <td>50 mM Tris-HCl, pH 7.9 
 50 mM NaCl</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>TSG10 buffer</td>
 <td>50 mM Tris-HCl, pH7.9 
 100 mM NaCl 
 10% v/v glycerol</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>TSG20 buffer</td>
 <td>50 mM Tris-HCl, pH8 
 100 mM NaCl 
 20% v/v glycerol</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>TSGD buffer</td>
 <td>50 mM Tris-HCl, pH7.9 
 100 mM NaCl 
 10% v/v glycerol 
 0.03% w/v DDM</td>
 <td>Store at 4 &deg;C and add DDM just before use</td>
 </tr>
 <tr>
 <td colspan="3">Table II. Solution recipes.</td>
 </tr>
 </tbody>
 </table>
 </td>
 </tr>
 </tbody>
 </table>

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

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 particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

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 are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

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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