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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 high-density lipoprotein (HDL) naturally occurring in blood. ApoA-1 is a 243 residue-long chain of short amphipathic α-helices and has a lipid-free soluble conformation. In vitro when in the presence of lipids, two copies of the protein ApoA-1 spontaneously rearrange to encircle the hydrophobic acyl chain portion of a lipid bilayer patch⁵. Engineered versions of ApoA-1 are generally called membrane scaffolding proteins (MSP), and an increasing number are commercially available as plasmids or as purified proteins. Repetitions or deletions of the α-helices in ApoA-1 result in longer⁶ or shorter⁷ membrane scaffolding proteins. This in turn makes it possible to form discs around 6 nm⁷ to 17 nm⁸ in diameter. There are different types of applications for the nanodiscs^3,9. The most commonly used application is to provide a near-native membrane environment for the stabilization of an integral membrane protein⁸, reviewed previously^3,9. A less-explored use is to provide a nanoscale membrane surface for the study of peripheral membrane proteins^{10,11,12,13,14,15,16,17}. Section 1 of the protocol below visualizes the procedure for making nanodiscs composed of phospholipids and membrane scaffolding protein.

Sample preparation is a bottleneck in most methods. Method-specific samples may add particular information, but they also make comparisons of results difficult. Therefore, it is simpler when samples are multimodal and can be used directly in several different methods. One advantage with the use of nanodiscs is the small size of the nanodisc in comparison to liposomes (e.g., the samples can be directly used for both TEM and non-denaturing gel electrophoresis, as in the present protocol).

Vesicles and liposomes have long been used to understand the function of membrane-interacting proteins. For structural studies and visualization, an example of the structural determination of a transmembrane protein in liposomes is available¹⁸. However, no high-resolution 3D structure of a monotopic membrane protein embedded on a liposome membrane has been published yet, as far as we know. Gold nanoparticles or antibodies can be used to visualize proteins binding to liposomes or vesicles using TEM¹⁹. Even though these probes are very specific, they might interfere with membrane-binding proteins by veiling the membrane binding site or by masking areas of interest with the flexible parts. Gold-labeled or antibody-complexed proteins could probably be analyzed on a gel, but this would increase the cost of the experiment.

Though liposomes are an excellent platform, one cannot be certain that the population has a particular ratio of protein per liposome, a feature that can be explored by the use of nanodiscs²⁰. In a liposome, cofactors and substrates can be trapped in the soluble interior. Substances that are membrane-soluble will share the same fate for both types of membrane mimetics. Nevertheless, as the bilayer area is smaller in nanodiscs, a smaller amount of substance is required to saturate the nanodisc membranes.

Understanding protein function through the determination of the atomic structure has been essential for many fields of research. Methods for protein structure determination include X-ray²¹; nuclear magnetic resonance (NMR)^22,23; and transmission electron microscopy (TEM)²⁴ at cryogenic temperatures, cryoEM. The resolution by cryoEM has lately been greatly improved, mainly due to the use of direct electron detectors^25,26. The macromolecules are imaged in thin, vitreous ice²⁷ in a near-native state. However, due to the low contrast of biological molecules, they become hard to detect in the size range of 100 - 200 kDa. For suitably sized samples, data collection can be made and the method of single particle reconstruction can be applied to obtain a structure²⁸.

However, the determination of protein structure by TEM is a multistep process. It usually starts with the evaluation of sample monodispersity by negative-stain TEM²⁹ using salts of heavy metals like phosphotungsten (PT)³⁰ or uranium³¹. Reconstruction of a low-resolution model of the negatively stained macromolecule is usually made and may yield important information on the molecular structure²⁹. In parallel, data collection using cryoEM may start. Care should be taken when evaluating negative-stain TEM data to avoid the misinterpretation of artefact formation. One particular artefact is the effect of the PT stain on phospholipids and liposomes³², resulting in the formation of long rods resembling stacks of coins viewed from the side³³. Such "rouleau" or "stacks" (hereafter denoted as "stacks") were observed early on for HDL³⁴, and later also for nanodiscs³⁵.

The stacking and reshaping of membranes may occur for many reasons. For example, it can be induced by co-factors like copper, shown by TEM imaging in an ammonium molybdate stain³⁶. A fraction of the membrane lipids in liposomes contained an iminodiacetic acid head group mimicking metal complexation by EDTA, thus stacking liposomes after the addition of copper ions³⁶. Stacking could also be due to a protein-protein interaction by a protein in or on the lipid bilayers (the stain used is not mentioned)³⁷. The stack formation of phospholipids by PT was observed early on; however, later work has focused on removing or abolishing this artifact formation³⁸.

Here, we propose a method to take advantage of the NaPT-induced nanodisc stacking for the study of membrane-binding proteins by TEM. In short, protein binding on the nanodiscs would prevent the nanodiscs from stacking. Though the reasons for the stacking are not clear, it was proposed³⁹ that there is an electrostatic interaction between the phospholipids and the phosphoryl group of PT, causing the discs to stick to each other (Figure 1A). The hypothesis behind our protocol is that when a protein binds to a nanodisc, most of the phospholipid surface is not available for the interaction with the PT due to steric hindrance by the protein. This would prevent stack formation (Figure 1B). Two conclusions can be drawn. First, the prevention of stacking means that the protein of interest has bound to the membrane. Secondly, the protein-ND complex can be treated with standard single-particle processing methods^24,40 to get a rough morphology of the complex. Furthermore, analyses by methods like non-denaturing gel electrophoresis or dynamic light scattering can be performed.

To demonstrate this hypothesis, we used the membrane-binding protein 5-lipoxygenase (5LO), which is involved in many inflammatory diseases^41,42. This 78-kDa protein requires calcium ions to bind to its membrane⁴³. Though this membrane association has been studied extensively using liposomes^44,45,46 and membrane fractions⁴⁷, these cannot be used for TEM analysis and structure determination.

The preparation of nanodiscs starts by mixing MSP with lipid resuspended in the detergent sodium cholate. After incubation on ice for 1 h, the detergent is slowly removed from the reconstitution mixture using an adsorbent resin. This kind of material is frequently made of polystyrene shaped into small beads. They are relatively hydrophobic and have a strong preference for binding detergent compared to lipids⁴⁸. After removing the hydrophobic beads and performing clarification using centrifugation, the nanodiscs are purified by size exclusion chromatography (SEC). The purified nanodiscs are mixed with a monotopic membrane protein (and possible cofactors) in an equimolar ratio (or several ratios for a titration) and are left to react (15 min). Analysis by TEM is carried out by applying µL-amounts of sample onto glow-discharged, carbon-coated grids and then by performing negative staining with NaPT. The same sample from when the aliquots were applied to the TEM grids can be used for analysis by non-denaturing or SDS PAGE gel-electrophoresis, as well as by various kinds of activity measurements, with no major changes.

Protokół

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 β-D-1-thiogalactopyranoside (IPTG) for 3 h at 18 °C.
   3. Prepare the lysis buffer (100 mM Tris-HCl, pH 8.0; 100 mM NaCl; and 10% glycerol). Add 1 mM TCEP immediately before use.
   4. After 3 h of induction at 18 °C, harvest the cells by centrifugation for 10 min at 4,500 x g and 4 °C. Discard the supernatant, weigh the harvested cells, and re-suspend them in lysis buffer at a ratio of 2 mL of lysis buffer per g of cells.
   5. Lyse the cells by pulsed sonication (repetition rate: 4 s ON, 4 s OFF) for 3 min at 80% amplitude.
   6. Centrifuge the lysates for 20 min at 49,000 x g and 4 °C. Discard the pellet from this centrifugation.
   7. Inject the supernatant into a 5-mL Ni⁺ chelating column connected to an automated liquid chromatography system preferably kept at 4 °C.
   8. Before the elution step (step 1.1.9), wash the column with buffers 1 - 3 below to remove all proteins except the His-tagged MSP. Monitor the protein content at 280 nm to follow the purification process; the UV recording at 280 nm should go back to baseline after each wash.
      1. Wash with wash buffer 1: 40 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, and 1% Triton, pH 8.0.
      2. Wash with wash buffer 2: 40 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, and 50 mM sodium cholate, pH 8.0.
      3. Wash with wash buffer 3: 40 mM Tris-HCl, 300 mM NaCl, and 50 mM imidazole, pH 8.0.
   9. Elute the MSP with 40 mM Tris-HCl, 300 mM NaCl, and 500 mM imidazole, pH 8.0.
   10. Change the buffer to MSP standard buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; and 0.5 mM EDTA) by gel filtration^8,35; the yield per batch should be around 7 mg L^-1.
2. Preparation of phospholipid stock solution
   1. Add 305 µL of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) dissolved in chloroform at a concentration of 25 mg/mL to a glass beaker and evaporate the chloroform by purging it with a gentle stream of nitrogen. Dry the lipid overnight in a vacuum desiccator. NOTE: This step is performed to remove the solvent from the lipid, which leaves a thin film of lipid at the bottom of the glass beaker.
   2. Resuspend the lipid cake in 200 µL of MSP standard buffer containing 100 mM sodium cholate by vortexing the tube until the solution becomes transparent; this will provide a solution with a POPC concentration of 50 mM.
      NOTE: In general, the molar ratio of lipid to detergent at this point should be 1:2 (lipid:detergent)⁸. This lipid-detergent mix can be stored at -80 °C for almost 2 months.
3. Preparation of hydrophobic beads for detergent removal
   1. Place 5 g of beads (dry-weight) in a 50-mL tube.
   2. Wash the beads with 30 mL of 100% methanol and then with 40 mL of ultrapure water.
   3. Wash the beads with 10 mL of MSP standard buffer. Finally, store the beads with 15 mL of MSP standard buffer at 4 °C.
4. Nanodisc reconstitution
   1. Dispense 190 µL of MSP1E3D1 (0.124 mM) into a microfuge tube and add 61.5 µL of phospholipid stock solution (50 mM POPC) to the same tube. Incubate the reconstitution mixture on wet ice for 1 h. NOTE: This equals a molar ratio of 1:130 (MSP1E3D1: POPC).
   2. Add hydrophobic beads at a concentration of 0.5 g of beads per mL of reconstitution mixture to initiate the self-assembly process. Incubate in a rotary incubator for 16 h at 4 °C.
   3. After incubation, centrifuge for 10 min at 13,000 x g and 4 °C to remove the precipitates and aggregates. Discard the pellet and save the supernatant.
   4. Equilibrate the size exclusion chromatography column (mounted on an automated liquid chromatography system) with MSP standard buffer until the absorbance at 280 nm is stable. Inject the supernatant into the column and collect the peak fractions.
   5. Measure the concentrations of nanodiscs in the peak fractions at 280 nm. Use the molar extinction coefficient of MSP1E3D1 (ε = 29,910 cm^-1 M^-1) for the calculation of the nanodisc concentration. NOTE: The number of lipids per nanodisc can be measured by a combination of radiolabeled lipids and phosphate analysis⁴ or by phosphate analysis alone.

2. Preparation of the Monotopic Protein 5-Lipoxygenase³⁵

1. Prepare an overnight starter culture. Supplement 50 mL of LB medium with 100 µg/mL ampicillin. Inoculate the medium with E. coli BL21 (DE3) containing the plasmid of the 5-lipoxygenase gene (ALOX5). Dilute the overnight starter culture in expression medium containing 42 mM Na₂HPO₄, 24 mM KH₂PO₄, 9 mM NaCl, 19 mM NH₄Cl, 1 mM MgSO₄, 0.1 mM CaCl₂, 0.2% D-glucose, 0.1%, 5 µM FeSO₄, and 100 µg/mL ampicillin. Grow the cells until the OD₆₀₀ is ~ 0.5 at 25 °C. Induce protein expression with 0.2 mM IPTG for 16 h at 20 °C³⁵.
2. Harvest by centrifugation for 10 min at 7,000 x g and 4 °C and discard the supernatant. Weigh the pellet found at the bottom of the tube containing the harvested cells and resuspend the harvested cells in lysis buffer (100 mM Tris-HCl, pH 8.0; 100 mM NaCl; 10% glycerol; and 1 mM TCEP) containing protease inhibitor and 0.5 mg/mL³⁵ lysozyme at a ratio of 2 mL of lysis buffer per g of cells.
3. Lyse the cells by sonication for 5 x 15 s at 80% amplitude. Remove the cell debris by centrifugation for 10 min at 7,000 x g and 4 °C. Perform ammonium sulfate precipitation to 30 - 60% saturation to precipitate the proteins in the solution³⁵. Centrifuge for 15 min at 16,000 x g and 4 °C. NOTE: The 5LO pellet can be rapidly frozen and stored at -80 °C for up to 6 months.
4. Resuspend the pellet with 20 mL of lysis buffer and centrifuge for 15 min at 40,000 x g and 4 °C.
5. Incubate the supernatant on an ATP agarose column at 4 °C for 30 min. Wash the column once with one column-volume of lysis buffer containing 0.5 M NaCl. Elute the 5LO with 20 mM ATP in lysis buffer containing 10 µM FeSO₄ and 20 µg/mL catalase. Perform gel filtration chromatography to remove the ATP³⁵.
   NOTE: The 5LO is unstable and should be used immediately after purification. Otherwise, it is recommended to halt at the step of ammonium sulphate precipitation (see the note after step 2.1.3).

3. Preparation of the Nanodisc-protein Complex

1. Prepare a total volume of 100 µL of complex. Mix 0.8 µM ND and 0.8 µM 5LO with the 1 mM Ca²⁺ present in the MSP standard buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 0.5 mM EDTA; and 1.5 mM CaCl₂) and incubate for 10 min on ice. NOTE: The sample can be stored for up to one month at 4 °C³⁵.

4. Analysis of the Samples

1. Gel electrophoresis
   1. Non-denaturing electrophoresis
      1. Mix 15 µL of the sample with 5 µL of loading buffer (50 mM BisTris, 6 N HCl, 50 mM NaCl, 10% (w/v) glycerol, and 0.001% Ponceau S, pH 7.2)⁴⁹ and load it on a 4 - 16% Bis-Tris gel to perform electrophoresis.
      2. Fill the cathode tank with light cathode buffer (50 mM Bis Tris; 50 mM Tricine, pH 6.8; and 0.002% Coomassie G-250) and the anode tank with running buffer (50 mM Bis Tris and 50 mM Tricine, pH 6.8). Start the separation by using a constant voltage at 150 V.
      3. Stop the electrophoretic separation when the Coomassie front reaches the end of the gel.
      4. Stain the gel with a standard Coomassie blue protocol.
   2. Denaturing electrophoresis
      1. Mix 40 µL of the sample with 10 µL of loading buffer containing SDS (0.05% (w/v) Bromophenol blue; 0.2 M Tris-HCl, pH 6.8; 20% (v/v) glycerol; 10% (w/v) SDS; and 10 mM 2-mercaptoethanol) and load it on a 4 - 20% Tris glycine gel to perform electrophoresis.
      2. Fill the cathode and anode tanks with running buffer (25 mM Tris-HCl, pH 6.8; 200 mM glycine; and 0.1% (w/v) SDS). Start the separation by using a constant voltage at 150 V.
      3. Stop the electrophoretic separation when the loading dye front reaches the end of the gel.
      4. Stain the gel with a standard Coomassie blue protocol.
2. Preparation of the NaPT solution
   1. Dissolve 1 g of phosphotungstate sodium salt in 50 mL of water by agitation at room temperature to give a 2% (w/v) acidic solution.
   2. Adjust the pH to 7.4 with 1 M NaOH. Remove the particles using a 0.22-µm syringe filter. Store the solution at room temperature or at 4 °C.
3. Preparation of the sample for TEM analysis
   1. Glow-discharge carbon-coated copper grids (400 mesh) for 20 s at 30 mA to render the grids hydrophilic before the adsorption of the sample. Place 3.5 µL of the sample (0.8 µM with respect to the nanodiscs) on the grid and incubate for 30 s. NOTE: The volume applied may vary between 2.5 and 5 µL, depending on the sample concentration. A suitable concentration range for nanodiscs is 0.5 - 1 µM.
   2. Blot off surplus solution using filter paper.
   3. Immediately stain the grid with a drop of 2% NaPT for 30 s. Blot off excess solution and leave the grid to air dry.
   4. Evaluate the grids by TEM. A microscope with 120-200 keV accelerating voltage suffices to estimate the extent of stacking. NOTE: For the present protocol, a calibrated transmission electron microscope was used, equipped with a 200-keV field emission gun.
   5. Record the TEM images. For the images showing long stacks, do not process further; images show the complex of nanodiscs, with extrinsic protein that may contain a few short stacks, but most particles are in the complex. Record several images and process these according to standard methods to obtain class-averages and a low-resolution, 3 dimensional model of the complex.
      NOTE: For the collection of negative-stain data, grids were made on at least three different days. For each day, fresh sample incubations (as in step 3.1) were made before the negative stain was applied.

Wyniki

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...

Dyskusje

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...

Materiały

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.