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Method Article

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

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DOI:

10.3791/55148

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March 5th, 2017

 ,  ,  , 
1Department of Biosciences and Nutrition, Karolinska Institutet, 2School of Technology and Health, KTH Royal Institute of Technology

In This Article

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 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 nanodiscs3,9. The most commonly used application is to provide a near-native membrane environment for the stabilization of an integral membrane protein8, reviewed previously3,9. A less-explored use is to provide a nanoscale membrane surface for the study of peripheral membrane proteins10,11,12,13,14,15,16,17. 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 detectors25,26. 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 methods24,40 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 diseases41,42. This 78-kDa protein requires calcium ions to bind to its membrane43. Though this membrane association has been studied extensively using liposomes44,45,46 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.

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Protocol

1. Preparation of Nanodiscs

  1. Expression and purification of the membrane scaffolding protein8,35
    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 filtration8,35; 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.

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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...

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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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Transmission electron microscope: JEOL2100FJEOL
CCD cameraTiez Video and Imaging Processing System GmbH, Germany
Glow dischargerBaltec
TEM grid: 400 meshTAABGM016/C
Size exclusion chromatography: Agilent SEC-5Agilent Technologies5190-2526
Superdex 200 HR 10/300GE Healthcare Life Sciences17-5172-01
Plasmid: MSP1E3D1Addgene20066
Bacteria: BL21DE3NEBC2527H
Bacteria: BL21 (DE3) T1R pRARE2Protein Science Facility, KI, Solna
Purification Matrix: ATP agaroseSigma AldrichA2767
Purification Matrix: HisTrap HP-5 mLGE Healthcare Life Sciences17-5247-01
Lipid: POPCAvanti polar lipids850457C25 mg/mL in chloroform
Hydrophobic beads: Bio-Beads, SM-2 ResinBio-Rad1523920
13 mm syringe filter: 0.2 μmPall life sciencesPN 4554T
Stain: Sodium phosphotungstate tribasic hydrateSigma Aldrich31648
2-mercaptoethanolSigma AldrichM3148-250ML
Sodium Dodecyl Sulfate (SDS)Bio-Rad161-0301
Protease inhibitor cocktailSigma Aldrich4693132001
TCEPSigma Aldrich646547
Detergent: Sodium cholate hydrateSigma AldrichC6445-10G
Sodium Cholate500 mM Sodium cholate. Resuspend in miliQ water and store at -20 °C.
Lipid Stock50 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 buffer20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5 mM EDTA.
Store at 4 °C.
Non-Denaturaing Electrophoresis Anode BufferThermo Fisher ScientificBN200150 mM Bis-Tris, 50 mM Tricine, pH 6.8
Non-Denaturaing Electrophoresis Cathode BufferThermo Fisher ScientificBN200250 mM Bis-Tris, 50 mM Tricine, pH 6.8, 0.002% Coomassie G-250
Non-Denaturaing Electrophoresis 4x Sample loading BufferThermo Fisher ScientificBN200350 mM Bis-Tris, pH 7.2, 6 N HCl, 50 mM NaCl, 10% (w/v) glycerol, 0.001% Ponceau S
Denaturaing Electrophoresis Running BufferIn-house recipe: 25 mM Tris-HCl, pH 6.8, 200 mM Glycine, 0.1% (w/v) SDS
Denaturaing Electrophoresis 5x Sample loading BufferIn-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 brothTryptone - 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.

References

  1. Kleinschmidt, J. H., Popot, J. L. Folding and stability of integral membrane proteins in amphipols. Arch Biochem Biophys. 564, 327-343 (2014).
  2. Frauenfeld, J., et al. A saposin-lipoprotein nanoparticle system for membrane proteins. Nat Methods. 13 (4), 345-351 (2016).
  3. Denisov, I. G., Sligar, S. G. Nanodiscs for structural and functional studies of membrane proteins. Nat Struct Mol Biol. 23 (6), 481-486 (2016).
  4. Bayburt, T. H., Grinkova, Y. V., Sligar, S. G. Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins. Nano Letters. 2 (8), 853-856 (2002).
  5. Jonas, A., Steinmetz, A., Churgay, L. The number of amphipathic alpha-helical segments of apolipoproteins A-I, E, and A-IV determines the size and functional properties of their reconstituted lipoprotein particles. J Biol Chem. 268 (3), 1596-1602 (1993).
  6. Grinkova, Y. V., Denisov, I. G., Sligar, S. G. Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers. Protein Eng Des Sel. 23 (11), 843-848 (2010).
  7. Hagn, F., Etzkorn, M., Raschle, T., Wagner, G. Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J Am Chem Soc. 135 (5), 1919-1925 (2013).
  8. Ritchie, T. K., et al. Methods in Enzymology. Duzgunes, N. ejat 464, Academic Press. 211-231 (2009).
  9. Schuler, M. A., Denisov, I. G., Sligar, S. G. Nanodiscs as a new tool to examine lipid-protein interactions. Methods Mol Biol. 974, 415-433 (2013).
  10. Nasr, M. L., et al. Membrane phospholipid bilayer as a determinant of monoacylglycerol lipase kinetic profile and conformational repertoire. Protein Sci. 22 (6), 774-787 (2013).
  11. Yokogawa, M., et al. NMR analyses of the interaction between the FYVE domain of early endosome antigen 1 (EEA1) and phosphoinositide embedded in a lipid bilayer. J Biol Chem. 287 (42), 34936-34945 (2012).
  12. Wan, C., et al. Insights into the molecular recognition of the granuphilin C2A domain with PI(4,5)P2. Chem Phys Lipids. 186, 61-67 (2015).
  13. Zhang, P., et al. An Isoform-Specific Myristylation Switch Targets Type II PKA Holoenzymes to Membranes. Structure. 23 (9), 1563-1572 (2015).
  14. Grushin, K., Miller, J., Dalm, D., Stoilova-McPhie, S. Factor VIII organisation on nanodiscs with different lipid composition. Thromb Haemost. 113 (4), 741-749 (2015).
  15. Baylon, J. L., Lenov, I. L., Sligar, S. G., Tajkhorshid, E. Characterizing the membrane-bound state of cytochrome P450 3A4: structure, depth of insertion, and orientation. J Am Chem Soc. 135 (23), 8542-8551 (2013).
  16. Mazhab-Jafari, M. T., et al. Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site. Proc Natl Acad Sci U S A. 112 (21), 6625-6630 (2015).
  17. Mazhab-Jafari, M. T., et al. Membrane-dependent modulation of the mTOR activator Rheb: NMR observations of a GTPase tethered to a lipid-bilayer nanodisc. J Am Chem Soc. 135 (9), 3367-3370 (2013).
  18. Wang, L., Sigworth, F. J. Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy. Nature. 461 (7261), 292-295 (2009).
  19. Ackerson, C. J., Powell, R. D., Hainfeld, J. F. Site-specific biomolecule labeling with gold clusters. Methods Enzymol. 481, 195-230 (2010).
  20. Boldog, T., Grimme, S., Li, M., Sligar, S. G., Hazelbauer, G. L. Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties. Proc Natl Acad Sci U S A. 103 (31), 11509-11514 (2006).
  21. Moraes, I., Evans, G., Sanchez-Weatherby, J., Newstead, S., Stewart, P. D. Membrane protein structure determination - the next generation. Biochim Biophys Acta. 1838 (1 Pt A), 78-87 (2014).
  22. Dias, D. M., Ciulli, A. NMR approaches in structure-based lead discovery: recent developments and new frontiers for targeting multi-protein complexes. Prog Biophys Mol Biol. 116 (2-3), 101-112 (2014).
  23. Viegas, A., Viennet, T., Etzkorn, M. The power, pitfalls and potential of the nanodisc system for NMR-based studies. Biol Chem. , (2016).
  24. Cheng, Y., Grigorieff, N., Penczek, P. A., Walz, T. A primer to single-particle cryo-electron microscopy. Cell. 161 (3), 438-449 (2015).
  25. Wu, S., Armache, J. P., Cheng, Y. Single-particle cryo-EM data acquisition by using direct electron detection camera. Microscopy (Oxf). 65 (1), 35-41 (2016).
  26. Li, X., et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods. 10 (6), 584-590 (2013).
  27. De Carlo, S., Adrian, M., Kalin, P., Mayer, J. M., Dubochet, J. Unexpected property of trehalose as observed by cryo-electron microscopy. J Microsc. 196 (1), 40-45 (1999).
  28. Nogales, E. The development of cryo-EM into a mainstream structural biology technique. Nat Methods. 13 (1), 24-27 (2016).
  29. Ohi, M., Li, Y., Cheng, Y., Walz, T. Negative Staining and Image Classification Powerful Tools in Modern Electron Microscopy. Biol Proced Online. 6, 23-34 (2004).
  30. Forte, T. M., Nordhausen, R. W. Electron microscopy of negatively stained lipoproteins. Methods Enzymol. 128, 442-457 (1986).
  31. Zhao, F. Q., Craig, R. Capturing time-resolved changes in molecular structure by negative staining. J Struct Biol. 141 (1), 43-52 (2003).
  32. Zhang, L., et al. Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy. J Lipid Res. 52 (1), 175-184 (2011).
  33. Zhang, L., et al. An optimized negative-staining protocol of electron microscopy for apoE4 POPC lipoprotein. J Lipid Res. 51 (5), 1228-1236 (2010).
  34. Matz, C. E., Jonas, A. Micellar complexes of human apolipoprotein A-I with phosphatidylcholines and cholesterol prepared from cholate-lipid dispersions. J Biol Chem. 257 (8), 4535-4540 (1982).
  35. Kumar, R. B., et al. Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs. PLoS One. 11 (3), e0152116(2016).
  36. Waggoner, T. A., Last, J. A., Kotula, P. G., Sasaki, D. Y. Self-assembled columns of stacked lipid bilayers mediated by metal ion recognition. J Am Chem Soc. 123 (3), 496-497 (2001).
  37. Kovacs, E., et al. Analysis of the Role of the C-Terminal Tail in the Regulation of the Epidermal Growth Factor Receptor. Mol Cell Biol. 35 (17), 3083-3102 (2015).
  38. Rames, M., Yu, Y., Ren, G. Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy. J Vis Exp. (90), e51087(2014).
  39. Zhang, L., Tong, H., Garewal, M., Ren, G. Optimized negative-staining electron microscopy for lipoprotein studies. Biochim Biophys Acta. 1830 (1), 2150-2159 (2013).
  40. Cong, Y., Ludtke, S. J. Single particle analysis at high resolution. Methods Enzymol. 482, 211-235 (2010).
  41. Radmark, O., Werz, O., Steinhilber, D., Samuelsson, B. 5-Lipoxygenase, a key enzyme for leukotriene biosynthesis in health and disease. Biochim Biophys Acta. 1851 (4), 331-339 (2015).
  42. Anwar, Y., Sabir, J. S., Qureshi, M. I., Saini, K. S. 5-lipoxygenase: a promising drug target against inflammatory diseases-biochemical and pharmacological regulation. Curr Drug Targets. 15 (4), 410-422 (2014).
  43. Radmark, O., Samuelsson, B. Regulation of the activity of 5-lipoxygenase, a key enzyme in leukotriene biosynthesis. Biochem Biophys Res Commun. 396 (1), 105-110 (2010).
  44. Noguchi, M., Miyano, M., Matsumoto, T., Noma, M. Human 5-lipoxygenase associates with phosphatidylcholine liposomes and modulates LTA4 synthetase activity. Biochim Biophys Acta. 1215 (3), 300-306 (1994).
  45. Pande, A. H., Qin, S., Tatulian, S. A. Membrane fluidity is a key modulator of membrane binding, insertion, and activity of 5-lipoxygenase. Biophys J. 88 (6), 4084-4094 (2005).
  46. Pande, A. H., et al. Modulation of human 5-lipoxygenase activity by membrane lipids. Biochemistry. 43 (46), 14653-14666 (2004).
  47. Wong, A., Hwang, S. M., Cook, M. N., Hogaboom, G. K., Crooke, S. T. Interactions of 5-lipoxygenase with membranes: studies on the association of soluble enzyme with membranes and alterations in enzyme activity. Biochemistry. 27 (18), 6763-6769 (1988).
  48. Rigaud, J. L., Levy, D., Mosser, G., Lambert, O. Detergent removal by non-polar polystyrene beads. European Biophysics Journal. 27 (4), 305-319 (1998).
  49. Wittig, I., Braun, H. P., Schagger, H. Blue native PAGE. Nat Protoc. 1 (1), 418-428 (2006).
  50. Rames, M., Yu, Y., Ren, G. Optimized negative staining: a high-throughput protocol for examining small and asymmetric protein structure by electron microscopy. J Vis Exp. (90), e51087(2014).
  51. Denisov, I. G., Grinkova, Y. V., Lazarides, A. A., Sligar, S. G. Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. J Am Chem Soc. 126 (11), 3477-3487 (2004).
  52. Hornschemeyer, P., Liss, V., Heermann, R., Jung, K., Hunke, S. Interaction Analysis of a Two-Component System Using Nanodiscs. PLoS One. 11 (2), 0149187(2016).
  53. Degrip, W. J., Vanoostrum, J., Bovee-Geurts, P. H. Selective detergent-extraction from mixed detergent/lipid/protein micelles, using cyclodextrin inclusion compounds: a novel generic approach for the preparation of proteoliposomes. Biochem J. 330 (Pt 2), 667-674 (1998).
  54. Martin, D. D., Budamagunta, M. S., Ryan, R. O., Voss, J. C., Oda, M. N. Apolipoprotein A-I assumes a "looped belt" conformation on reconstituted high density lipoprotein. J Biol Chem. 281 (29), 20418-20426 (2006).
  55. Cerione, R. A., Ross, E. M. Reconstitution of receptors and G proteins in phospholipid vesicles. Methods Enzymol. 195, 329-342 (1991).
  56. Shaw, A. W., McLean, M. A., Sligar, S. G. Phospholipid phase transitions in homogeneous nanometer scale bilayer discs. FEBS Lett. 556 (1-3), 260-264 (2004).
  57. Meer, G., Voelker, D. R., Feigenson, G. W. Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol. 9 (2), 112-124 (2008).
  58. Civjan, N. R., Bayburt, T. H., Schuler, M. A., Sligar, S. G. Direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers. Biotechniques. 35 (3), 553-562 (2003).
  59. 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(2012).
  60. Brooks, S. P., Storey, K. B. Bound and determined: a computer program for making buffers of defined ion concentrations. Anal Biochem. 201 (1), 119-126 (1992).
  61. Gilbert, N. C., et al. The structure of human 5-lipoxygenase. Science. 331 (6014), 217-219 (2011).
  62. Radmark, O. 5-lipoxygenase-derived leukotrienes: mediators also of atherosclerotic inflammation. Arterioscler Thromb Vasc Biol. 23 (7), 1140-1142 (2003).
  63. Gao, Y., Cao, E., Julius, D., Cheng, Y. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature. 534 (7607), 347-351 (2016).
  64. Bayburt, T. H., Sligar, S. G. Self-assembly of single integral membrane proteins into soluble nanoscale phospholipid bilayers. Protein Sci. 12 (11), 2476-2481 (2003).

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Nanodisc PreparationMembrane Protein BindingNegative Stain TEMSize Exclusion ChromatographyNon-denaturing Gel ElectrophoresisPhosphotungstate StainingMonotopic Protein AnalysisNanodisc StackingProtein Nanodisc Complex
Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy
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