Name: method to visualize and analyze membrane interacting proteins by transmission electron microscopy
Uploaded: 2017-03-05T14:00:00
Description: Watch this Scientific Journal Video about Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy at JoVE.com

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 Sj&#246;berg for sharing their technical expertise and for their timely assistance.

1. Preparation of Nanodiscs
<ol>
	<li>Expression and purification of the membrane scaffolding protein8,35
	<ol>
		<li>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 &#181;g/mL Kanamycin at 37 &#176;C. Dilute the overnight starter culture in 2 L of terrific broth medium supplemented with 50 &#181;g/mL kanamycin.</li>
		<li>Grow the cells...

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...

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 &#945;-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 &#945;-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 &#34;rouleau&#34; or &#34;stacks&#34; (hereafter denoted as &#34;stacks&#34;) 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 &#181;L-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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Transmission electron microscope: JEOL2100F</td>
			<td>JEOL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Tiez Video and Imaging Processing System GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glow discharger</td>
			<td>Baltec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TEM grid: 400 mesh</td>
			<td>TAAB</td>
			<td>GM016/C</td>
			<td></td>
		</tr>
		<tr>
			<td>Size exclusion chromatography: Agilent SEC-5</td>
			<td>Agilent Technologies</td>
			<td>5190-2526</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 HR 10/300</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-5172-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid:MSP1E3D1</td>
			<td>Addgene</td>
			<td>20066</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteria: BL21DE3</td>
			<td>NEB</td>
			<td>C2527H</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteria: BL21 (DE3) T1R pRARE2</td>
			<td>Protein Science Facility, KI, Solna</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Purification Matrix: ATP agarose</td>
			<td>Sigma Aldrich</td>
			<td>A2767</td>
			<td></td>
		</tr>
		<tr>
			<td>Purification Matrix: HisTrap HP-5 ml</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-5247-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid:POPC</td>
			<td>Avanti polar lipids</td>
			<td>850457C</td>
			<td>25 mg/ml in chloroform</td>
		</tr>
		<tr>
			<td>Hydrophobic beads: Bio-Beads, SM-2 Resin</td>
			<td>Bio-Rad</td>
			<td>1523920</td>
			<td></td>
		</tr>
		<tr>
			<td>13 mm syringe filter: 0.2 &#956;m</td>
			<td>Pall life sciences</td>
			<td>PN 4554T</td>
			<td></td>
		</tr>
		<tr>
			<td>Stain: Sodium phosphotungstate tribasic hydrate</td>
			<td>Sigma Aldrich</td>
			<td>31648</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Bio-Rad</td>
			<td>161-0301</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma Aldrich</td>
			<td>4693132001</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Sigma Aldrich</td>
			<td>646547</td>
			<td></td>
		</tr>
		<tr>
			<td>Detergent: Sodium cholate hydrate</td>
			<td>Sigma Aldrich</td>
			<td>C6445-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cholate</td>
			<td>500 mM Sodium cholate</td>
			<td></td>
			<td>Resuspend in miliQ water and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Lipid Stock</td>
			<td>50 mM POPC, 100 mM sodium cholate, 20 mM Tris-HCl pH 7.5, 100 mM NaCl</td>
			<td></td>
			<td>Store at 4&#176;C for a week or 
			Store -80&#176;C for a month, after purging the solution with nitrogen</td>
		</tr>
		<tr>
			<td>MSP standard buffer</td>
			<td>20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mMEDTA</td>
			<td></td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Non-Denaturaing Electrophoresis Anode Buffer</td>
			<td>50 mM Bis Tris 50 mM Tricine, pH 6.8</td>
			<td>BN2001</td>
			<td>Purchased from Thermofisher Scientific</td>
		</tr>
		<tr>
			<td>Non-Denaturaing Electrophoresis Cathode Buffer</td>
			<td>50 mM Bis Tris 50 mM Tricine, pH 6.8 0.002% Coomassie G-250</td>
			<td>BN2002</td>
			<td>Purchased from Thermofisher Scientific</td>
		</tr>
		<tr>
			<td>Non-Denaturaing Electrophoresis 4X Sample loading Buffer</td>
			<td>50 mM BisTrispH 7.2, 6N HCl, 50 mM NaCl, 10% (w/v) glycerol, 0.001% Ponceau S</td>
			<td>BN2003</td>
			<td>Purchased from Thermofisher Scientific</td>
		</tr>
		<tr>
			<td>Denaturaing Electrophoresis Running Buffer</td>
			<td>25 mM Tris-HCl pH 6.8, 200 mM Glycine, 0.1 % (w/v) SDS</td>
			<td></td>
			<td>Inhouse receipe</td>
		</tr>
		<tr>
			<td>Denaturaing Electrophoresis 5X Sample loading Buffer</td>
			<td>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</td>
			<td></td>
			<td>Inhouse receipe</td>
		</tr>
		<tr>
			<td>Terrific broth</td>
			<td>Tryptone - 12.0g 
			Yeast Extract - 24.0g 
			100 mL 0.17M KH2PO4 and 0.72M K2HPO4 
			Glycerol - 4 mL</td>
			<td></td>
			<td>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</td>
		</tr>
	</tbody>
</table>

method to visualize and analyze membrane interacting proteins by transmission electron microscopy

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.

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 &#34;empty nanodiscs&#34; (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...

Watch this Scientific Journal Video about Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy at JoVE.com

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

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.

Метод визуализации и анализа Мембранные белков, взаимодействующих с помощью просвечивающей электронной микроскопии

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

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