Department of Biochemistry and Molecular Biology, University of British Columbia
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Bao, H., Duong, F., Chan, C. S. A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles. J. Vis. Exp. (66), e3910, doi:10.3791/3910 (2012).
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.
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 MalFGK2 complex and the surface of the MSP amphipathic protein. The final product is a discoid particle made of two molecules of MSP wrapping around the MalFGK2 complex. The particles are separated from the adducts and aggregates by ultra-centrifugation and analytical size-exclusion chromatography. The particles are characterized by native-gel electrophoresis and dynamic light scattering spectroscopy.
1. Preparation of the Membrane Scaffold Protein, MSP
2. Preparation of the MalFGK2 Complex
3. Preparation of Phospholipids
4. Preparation of Bio-Beads
5. Nanodisc Reconstitution
6. Native Gel Electrophoresis
7. Dynamic Light Scattering (DLS)
8. ATPase Measurements
9. Representative Results
The nanodiscs are purified by gel filtration chromatography (Figure 2A, left). The chromatogram shows that the majority of the reconstituted discs (black trace) elute as a single peak, whereas discs made with excess lipids (red trace) elute in the void volume and as a series of broad peaks. The quality of the nanodiscs is further analyzed by native gel electrophoresis and dynamic light scattering spectroscopy (DLS). Properly reconstituted discs migrate as a sharp band on the gel whereas those reconstituted in the presence of an excess of lipids migrate as a smear (Figure 2A, right). Analysis by DLS shows that the disc population is homogeneous with an average diameter of 11.4 nm (Figure 2B). The reconstituted discs have an apparent molecular weight of 215 kDa based on the DLS approximation. Samples reconstituted in the presence of excess lipids display widely distributed radii around 100 nm, which is typical for non-homogeneous samples.
The quality of the MalFGK2 complex is assessed by native-gel electrophoresis and its activity by ATPase measurements (Figure 3). The maltose binding protein MalE binds with a high-affinity to the MalFGK2 transporter9, 10. Using non-denaturing gel electrophoresis, it is possible to detect a complex between MalE and MalFGK2 (Figure 3A). The stimulation of the MalK2 ATPase activity by MalE is shown in Figure 3B.
Figure 1. Typical flowchart for the reconstitution protocol.
Figure 2. Quality control of the nanodisc preparation. A. Gel filtration analysis (Superdex 200 HR 10/300 column) of the nanodiscs reconstituted at low lipid ratio (1/3/60; black trace) or high lipid ratio (1/3/400; red trace). Native gel electrophoresis of the same disc preparation. Molecular weight markers in kDa are indicated. B. Dynamic light scattering analysis of the same disc preparation. Click here to view larger figure.
Figure 3. Analysis of the MalFGK2-nanodisc particles. A. Gel shift analysis of MalE incubated with increasing amounts of MalFGK2-nanodisc particles. B. ATPase activity of the MalFGK2-nanodisc particles as a function of MalE concentration.
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 change conformations during the transport cycle but the contribution of lipids to these conformational changes remain to be explored.
The making of the nanodisc is relatively simple, yet small deviations from optimal conditions can lead to irreversible protein aggregation. In this report, we stress the importance of choosing a correct protein:lipid ratio because an excess amount of lipids will lead to the production of large polydisperse liposome-like particles that do not respond to the biophysical qualification of a nanodisc. A previous analysis employed very high MSP:lipids ratio for the reconstitution of the maltose transporter(1/120 compared to 1/20 in our analysis)but the particles formed were not further characterized11. In any case, it is crucial to carefully analyze the quality of the reconstituted material using techniques other than gel filtration, such as light scattering spectroscopy, analytical ultracentrifugation or more simply, native gel electrophoresis. The importance of this quality control was highlighted during the reconstitution of bacteriorhodopsin and P-glycoprotein12. Two other parameters can greatly affect the success of the reconstitution: 1) the purity of the target membrane protein and the absence of aggregates and 2) the correct ratio between membrane protein and scaffold protein. In addition, the type of phospholipid incorporated into the disc, as well as the length of the membrane scaffold protein, can contribute to the efficiency of the reconstitution. Other parameters such as the solubility and stability of the membrane protein in detergent solution, the buffer system, the temperature and time-length of the reconstitution may also need to be adjusted when optimizing the reconstitution protocol. For example, membrane scaffold proteins of different lengths produce nanodiscs of different sizes1, which can help the reconstitution of larger membrane protein complexes or membrane protein oligomers.
The nanodisc has been successfully combined with biophysical methods such SPR, ITC, and fluorescence spectroscopy12-16 to help membrane research that is struggling with quantitative methodology. Recently, the nanodisc has been combined with quantitative mass spectrometry to help identifying membrane protein interactomes with better coverage and accuracy17. The pharmaceutical industry has been limited by the same difficulties that plague academia in studying membrane proteins, although it is fact that the most frequently prescribed drugs target membrane proteins (receptors, channels, transporters, sensors). It can be predicted that the nanodisc will help development of drug screening strategies that work with membrane embedded proteins.
No conflicts of interest declared.
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.
|Amicon Ultra-4 50K centrifugal filter||Millipore||UFC805008||Follow manufacturer’s protocol for proper use|
|Bio-Beads SM-2 Adsorbent||Bio-Rad||152-3920|
|E. coli total lipids||Avanti Polar Lipids||100500C||Dissolved in chloroform, handle as appropriate for an organic solvent|
|Ni sepharose HP resin||GE Healthcare||17-5268-01|
|Phosphorous standard solution||Sigma-Aldrich||P3869|
|Superdex 200 HR 10/300||GE Healthcare||17-5172-01|
|Table I. Specific reagents.|