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Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.
Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.
In medical research, much attention is focused on membrane proteins, either intrinsic or extrinsic, involved in a variety of lipid interactions. Working with lipid-interacting proteins includes either selecting a substitute to the lipids, such as detergents, amphipols1, or small proteins2, or finding a membrane substitute that keeps the protein soluble and active. Lipoic membrane substitutes include liposomes and nanodiscs (ND)3,4.
Nanodiscs are near-native membrane platforms developed by engineering the protein part, ApoA-1, of the hi....
1. Preparation of Nanodiscs
The method we propose depends upon the preparation of nanodiscs to provide the membrane surface for monotopic membrane-protein binding. As there is no transmembrane protein embedded into the nanodisc lipid bilayer, the nanodiscs are here denoted as "empty nanodiscs" (Figure 2A). These have a calculated molecular weight of 256 kDa for a composition of two MSP1E3D1 scaffolding proteins and around 260 molecules of POPC8. Using this protein:lip.......
The method can be separated into three parts: the reconstitution of empty nanodiscs, the preparation of protein-nanodisc complexes, and the negative staining for the TEM of these complexes. Each part will be addressed separately regarding limitations of the technique, critical steps, and useful modifications.
Reconstitution of empty nanodiscs. Critical steps and limitations in the production and use of nanodiscs.
For the preparation of the empty nanodiscs, it is essent.......
The authors thank the Swedish Research Council, Stockholm County Council, and KI funds for their support. The expression and purification of MSP was performed at the Karolinska Institutet/SciLifeLab Protein Science Core Facility (http://PSF.ki.se). The authors would also like to thank Dr. Pasi Purhonen and Dr. Mathilda Sjöberg for sharing their technical expertise and for their timely assistance.
....Name | Company | Catalog Number | Comments |
Transmission electron microscope: JEOL2100F | JEOL | ||
CCD camera | Tiez Video and Imaging Processing System GmbH, Germany | ||
Glow discharger | Baltec | ||
TEM grid: 400 mesh | TAAB | GM016/C | |
Size exclusion chromatography: Agilent SEC-5 | Agilent Technologies | 5190-2526 | |
Superdex 200 HR 10/300 | GE Healthcare Life Sciences | 17-5172-01 | |
Plasmid:MSP1E3D1 | Addgene | 20066 | |
Bacteria: BL21DE3 | NEB | C2527H | |
Bacteria: BL21 (DE3) T1R pRARE2 | Protein Science Facility, KI, Solna | ||
Purification Matrix: ATP agarose | Sigma Aldrich | A2767 | |
Purification Matrix: HisTrap HP-5 ml | GE Healthcare Life Sciences | 17-5247-01 | |
Lipid:POPC | Avanti polar lipids | 850457C | 25 mg/ml in chloroform |
Hydrophobic beads: Bio-Beads, SM-2 Resin | Bio-Rad | 1523920 | |
13 mm syringe filter: 0.2 μm | Pall life sciences | PN 4554T | |
Stain: Sodium phosphotungstate tribasic hydrate | Sigma Aldrich | 31648 | |
2-mercaptoethanol | Sigma Aldrich | M3148-250ML | |
Sodium Dodecyl Sulfate (SDS) | Bio-Rad | 161-0301 | |
Protease inhibitor cocktail | Sigma Aldrich | 4693132001 | |
TCEP | Sigma Aldrich | 646547 | |
Detergent: Sodium cholate hydrate | Sigma Aldrich | C6445-10G | |
Sodium Cholate | 500 mM Sodium cholate | Resuspend in miliQ water and store at -20°C | |
Lipid Stock | 50 mM POPC, 100 mM sodium cholate, 20 mM Tris-HCl pH 7.5, 100 mM NaCl | Store at 4°C for a week or Store -80°C for a month, after purging the solution with nitrogen | |
MSP standard buffer | 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mMEDTA | Store at 4°C | |
Non-Denaturaing Electrophoresis Anode Buffer | 50 mM Bis Tris 50 mM Tricine, pH 6.8 | BN2001 | Purchased from Thermofisher Scientific |
Non-Denaturaing Electrophoresis Cathode Buffer | 50 mM Bis Tris 50 mM Tricine, pH 6.8 0.002% Coomassie G-250 | BN2002 | Purchased from Thermofisher Scientific |
Non-Denaturaing Electrophoresis 4X Sample loading Buffer | 50 mM BisTrispH 7.2, 6N HCl, 50 mM NaCl, 10% (w/v) glycerol, 0.001% Ponceau S | BN2003 | Purchased from Thermofisher Scientific |
Denaturaing Electrophoresis Running Buffer | 25 mM Tris-HCl pH 6.8, 200 mM Glycine, 0.1 % (w/v) SDS | Inhouse receipe | |
Denaturaing Electrophoresis 5X Sample loading Buffer | 0.05 % (w/v) Bromophenolblue, 0.2 M Tris-HCl pH 6.8, 20 % (v/v) glycerol, 10% (w/v) SDS,10 mM 2-mercaptoethanol | Inhouse receipe | |
Terrific broth | Tryptone - 12.0g Yeast Extract - 24.0g 100 mL 0.17M KH2PO4 and 0.72M K2HPO4 Glycerol - 4 mL | Tryptone, yeast extract and glycerol were prepared to 900 ml and autoclaved seperately. KH2PO4 and K2HPO4 were prepared and autoclaved separately. Both were mixed before using the medium |
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