Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Preparation of Nanodiscs

1. Expression and purification of the membrane scaffolding protein^8,35
   1. Express the His-tagged MSP1E3D1 in the E. coli BL21 (DE3) T1R pRARE2 strain in flasks. Prepare a 50-mL overnight starter culture with LB medium supplemented with 50 µg/mL Kanamycin at 37 °C. Dilute the overnight starter culture in 2 L of terrific broth medium supplemented with 50 µg/mL kanamycin.
   2. Grow the cells at 37 °C until the optical density at 600 nm (OD₆₀₀) reaches approximately 3. Induce the protein expression with 0.5 mM isopropyl ....

Results

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:lipid ratio.......

Discussion

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.......

Materials

List of materials used in this article

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 mM EDTA. Store at 4 °C.
Non-Denaturaing Electrophoresis Anode Buffer	Thermo Fisher Scientific	BN2001	50 mM Bis-Tris, 50 mM Tricine, pH 6.8
Non-Denaturaing Electrophoresis Cathode Buffer	Thermo Fisher Scientific	BN2002	50 mM Bis-Tris, 50 mM Tricine, pH 6.8, 0.002% Coomassie G-250
Non-Denaturaing Electrophoresis 4x Sample loading Buffer	Thermo Fisher Scientific	BN2003	50 mM Bis-Tris, pH 7.2, 6 N HCl, 50 mM NaCl, 10% (w/v) glycerol, 0.001% Ponceau S
Denaturaing Electrophoresis Running Buffer			In-house recipe: 25 mM Tris-HCl, pH 6.8, 200 mM Glycine, 0.1% (w/v) SDS
Denaturaing Electrophoresis 5x Sample loading Buffer			In-house recipe: 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
Terrific broth			Tryptone - 12.0 g, Yeast Extract - 24.0 g, 100 mL 0.17 M KH2PO4 and 0.72 M 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.