This study was supported by the Velux Stiftung (A.K.M.), NIH grant EY024207 (A.K.M.) and the Austrian Science Fund (FWF) project J3686 (B.P.).

1. Preparation of Liposomes and Proteoliposomes
<ol>
	<li>Liposome Formation
	<ol>
		<li>Using a glass syringe, add 1,435 &#181;l POPC (25 mg/ml, in chloroform) and 160 &#181;l POPG (25 mg/ml, in chloroform) to a round bottom flask to obtain 52.5 &#181;mol lipids in a molar ratio of POPC:POPG = 9:1.</li>
		<li>Dry the lipids for 30 min using a rotary evaporator at a rotation speed of 145 rpm (no water bath is needed for this volume of solvent), then transfer the flask to a vacuum desiccator for at least 3 hr, or overni...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MurJ+and+a+novel+lipid+II+flippase+are+required+for+cell+wall+biogenesis+in+Bacillus+subtilis.">MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. 112, 6437-6442 (2015).</li><li>Ruiz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Flippases+for+Bacterial+Peptidoglycan+Biosynthesis.">Lipid Flippases for Bacterial Peptidoglycan Biosynthesis.</a> Lipid Insights. 8, 21-31 (2015).</li><li>Rick, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+the+wzxE+gene+of+Escherichia+coli+K-12+encodes+a+protein+involved+in+the+transbilayer+movement+of+a+trisaccharide-lipid+intermediate+in+the+assembly+of+enterobacterial+common+antigen.">Evidence that the wzxE gene of Escherichia coli K-12 encodes a protein involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen.</a> J. Biol. Chem. 278, 16534-16542 (2003).</li><li>Suzuki, J., Imanishi, E., Nagata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+of+phosphatidylserine+by+Xk-related+protein+family+members+during+apoptosis.">Exposure of phosphatidylserine by Xk-related protein family members during apoptosis.</a> J. Biol. 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The scramblase activity assay enabled us originally to determine that opsin has phospholipid scramblase activity 2. The assay also allowed us to characterize opsin&#39;s scramblase activity by testing specificity (we used a variety of NBD-labeled reporter lipids such as NBD-phosphatidylethanolamine, labeled with NBD on an acyl chain as shown for NBD-PC in Figure 1A, or on the headgroup, NBD-sphingomyelin or NBD- phosphatidylserine 2), the effect of vesicle lipid composition (e.g...

The photoreceptor rhodopsin, a prototypical G protein-coupled receptor (reviewed for example in reference 1), is the first phospholipid scramblase to be identified and biochemically verified 2,3. Scramblases are phospholipid transporters that increase the intrinsically slow rate of transbilayer phospholipid movement to physiologically appropriate levels in a bidirectional, ATP-independent manner 4-6. Examples of their actions can be found in the endoplasmic reticulum and bacterial cytoplasmic membrane where constitutive scrambling is needed for membrane homeostasis and growth, as well as for a variety of glycosylation pathways 5. Regulated phospholipid scrambling is needed to expose phosphatidylserine (PS) on the surface of apoptotic cells where it acts as an &#34;eat-me&#34;-signal for macrophages 7 and provides a procoagulant surface on activated blood platelets to catalyze the production of protein factors needed for blood clotting. In photoreceptor disc membranes, rhodopsin&#39;s scrambling activity has been suggested to counteract the phospholipid imbalance between the two membrane leaflets of the bilayer that is generated by the ATP-dependent, unidirectional lipid flippase ABCA4 4,8,910-12.
Despite the physiological importance of scramblases, their identity remained elusive until rhodopsin was reported as a scramblase in photoreceptor discs 2, members of the TMEM16 protein family were identified as Ca2+-dependent scramblases needed for PS exposure at the plasma membrane (reviewed in reference 13), and the bacterial protein FtsW was proposed as a Lipid II scramblase required for peptidoglycan synthesis 14. These discoveries were based on the reconstitution of purified proteins in liposomes and demonstration of scramblase activity in the resulting proteoliposomes using the methodology described here. Other potential scramblases 15-21&#160;&#8212; the MurJ and AmJ proteins implicated in peptidoglycan biosynthesis, WzxE and related proteins implicated in scrambling O-antigen precursors, MprF protein needed to translocate aminoacylated phosphatidylglycerol across the bacterial cytoplasmic membrane, and Xkr8 family members that have been proposed to expose PS on the surface of apoptotic cells&#160;&#8212; remain to be tested biochemically. This highlights the importance of a robust assay to identify and characterize scramblase activity.
Here, we describe the reconstitution of purified opsin, the apoprotein of the photoreceptor rhodopsin, into large unilamellar vesicles (LUVs), and subsequent analysis of scramblase activity in the resulting proteoliposomes using a fluorescence-based assay. There are several well-described protocols available in the literature for the heterologous expression and purification of opsin, therefore we will not describe it in this protocol; we use the protocols described in Goren et al. 3 which yields FLAG-tagged, thermostable opsin at about 100 ng/&#181;l in 0.1% (w/v) dodecylmaltoside (DDM).
Reconstitution is achieved by treating LUVs with sufficient detergent so that they swell but do not dissolve. Under these conditions, a membrane protein&#160;&#8212; supplied in the form of protein-detergent micelles&#160;&#8212; will integrate into the liposomes and become reconstituted into the liposome membrane upon detergent removal, resulting in proteoliposomes. To reconstitute opsin (obtained as a purified protein in 0.1% (w/v) DDM), LUVs are prepared from a mixture of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) and saturated with DDM before adding opsin and NBD-PC. The detergent is then removed by treating the sample with polystyrene beads.
The principle underlying the fluorescence-based assay is shown in Figure 1B. LUVs are symmetrically reconstituted with a trace amount of NBD-PC or other NBD-labeled fluorescent phospholipid reporter (Figure 1A). On adding dithionite, a membrane-impermeant dianion, NBD-PC molecules in the outer leaflet of the LUVs are rendered non-fluorescent as the nitro-group of NBD is reduced to a non-fluorescent amino-group. As neither NBD-PC molecules nor dithionite are able to traverse the membrane on the time-scale of the experiment (&#60;10 min), this results in 50% reduction of the fluorescent signal. However, if the liposomes are reconstituted with a scramblase, NBD-PC molecules in the inner leaflet can scramble rapidly to the outside where they are reduced. This results in the total loss of fluorescence in the ideal case (Figure 1C).
<img alt="Figure 1" src="/files/ftp_upload/54635/54635fig1.jpg" /> 
Figure 1: Schematic representation of the scramblase activity assay. The assay uses a fluorescent NBD-labeled reporter lipid; NBD-PC is shown (A). Large unilamellar vesicles are reconstituted with a trace amount of NBD-PC. Reconstitution produces symmetric vesicles, with NBD-PC distributed equally in the outer and inner leaflets. Dithionite (S2O42-) chemically reduces the nitro-group of NBD to a non-fluorescent amino-group. Treatment of protein-free liposomes with dithionite (B, top) causes a 50% reduction of fluorescence since only the NBD-PC molecules in the outer leaflet are reduced: dithionite is negatively charged and cannot cross the membrane to react with NBD-PC molecules in the inner leaflet. Dithionite treatment of opsin-containing proteoliposomes (B, bottom), i.e., scramblase-active proteoliposomes, results in 100% loss of fluorescence as opsin facilitates movement of NBD-PC between the inner and the outer leaflet. (C) shows idealized fluorescence traces obtained on treating protein-free liposomes and opsin-containing proteoliposomes with dithionite. The rate of fluorescence loss is the same in both cases indicating that the chemical reduction of NBD by dithionite is rate-limiting, and that scrambling occurs at a rate equal to or greater than the rate of the chemical reaction. Traces obtained from an actual experiment are shown in Figure 3. <a href="https://www.jove.com/files/ftp_upload/54635/54635fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The methods we describe can be used to reconstitute and assay other purified proteins, as well as mixtures of membrane proteins obtained, for example, by extracting microsomes with detergent 22.

<table border="0" cellpadding="0" cellspacing="0" width="1148">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:141px;">Avanti Polar Lipids</td>
			<td style="width:132px;">850457C</td>
			<td style="width:193px;">POPC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>840457C</td>
			<td>POPG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-an-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>810130C</td>
			<td>NBD-PC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td>
			<td>VWR Scientific</td>
			<td>EM-5330</td>
			<td>HEPES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma</td>
			<td>S7653-1KG</td>
			<td>NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dodecyl-&#946;-d-maltoside&#160;</td>
			<td>Anatrace</td>
			<td>D310 5 GM</td>
			<td>DDM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorimeter cuvettes</td>
			<td>sigma</td>
			<td>C0918-100EA</td>
			<td>cuvettes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectrofluorometer</td>
			<td colspan="2">Photon Technology International, Inc.</td>
			<td>fluorimeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydrosulfite technical grade, 85%</td>
			<td>Sigma</td>
			<td>157953-5G</td>
			<td>dithionite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism 5 software</td>
			<td></td>
			<td></td>
			<td>Prism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>VWR</td>
			<td>JTX171-3</td>
			<td>Tris</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIPEX 10 mL extruder&#160;</td>
			<td>Northern Lipids, Inc.</td>
			<td></td>
			<td>Extruder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman, Drain disc, PE, 25 mm</td>
			<td>Sigma</td>
			<td style="width:132px;">28156-243</td>
			<td>Disc support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman Nuclepore Track-Etched Membranes, 0.4 &#181;m, 25 mm diameter</td>
			<td>Sigma</td>
			<td style="width:132px;">WHA110607</td>
			<td>400 nm membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman Nucleopore Track-Etched Membranes, 0.2 &#181;m, 25 mm diameter</td>
			<td>Sigma</td>
			<td style="width:132px;">WHA110606</td>
			<td>200 nm membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium phosphate</td>
			<td>Sigma</td>
			<td>S3264-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VWR Culture Tubes, Disposable, Borosilicate Glass, 13x100 mm</td>
			<td>VWR Scientific</td>
			<td>47729-572</td>
			<td>glass tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perchloric acid</td>
			<td>Sigma</td>
			<td>30755-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Molybdate Tetrahydrate</td>
			<td>Sigma</td>
			<td>A-7302</td>
			<td>ammonium molybdate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(+)-Sodium L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25G</td>
			<td>sodium ascorbate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bio-Beads SM2 adsorbents</td>
			<td>Bio Rad</td>
			<td>1523920</td>
			<td>polystyrene beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;2.0 mL Microtubes clear</td>
			<td>VWR Scientific</td>
			<td>10011-742</td>
			<td>Reconstitution tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reconstitution glass tube</td>
			<td>VWR Scientific</td>
			<td style="width:132px;">53283-800</td>
			<td>Reconstitution glass tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer&#160;</td>
			<td>Malvern&#160;</td>
			<td></td>
			<td>DLS</td>
		</tr>
	</tbody>
</table>

a fluorescence-based assay of phospholipid scramblase activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe the reconstitution of opsin into LUVs to characterize its scramblase activity using a fluorescence-based assay. We analyze the results to place a lower limit on the rate of opsin-mediated phospholipid scrambling and to determine the oligomeric state in which opsin functionally reconstitutes into the vesicles.
To identify optimal reconstitution conditions, it is necessary to determine empirically the amount of deterge...

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A Fluorescence-based Assay of Phospholipid Scramblase Activity

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

The overall goal of this procedure is to reconstitute a typical G protein coupled receptor into large unilamellar vesicles, and to measure it's ability to carry out 80p independent translocation of phospholipids across a lipid bilayer using a flurorescence-based assay. This method can help answer key questions in the inter-cellular lipid transport field, such as what is the molecular identity of proteins that can scramble off liplipids and what is their mechanism. The main advantage of this technique is that it is very reliable and robust.Once your reconstitution procedure is established for a given lipid composition and a preferred detergent. To begin this procedure, use a glass syringe to add 1435 microliters of a 25 milligram per milliliter solution of POPC and chloroform to a round bottom flask. Add 160 microliters of a 25 milligram per milliliter solution of POPG and chloroform to obtain 52.5 micro-malipids in a nine to one molar ratio of POPC to POPG.Next, dry the lipids for 30 minutes using a rotary evaporator set to 145 RPM. Once drying is complete, transfer the flask to a vacuum desiccator for three hours at room temperature. Then, add 10 milliliters of buffer A to hydrate the died lipid film.Gently swirl the flask until a homogeneous turbid suspension is formed. Sonicate the suspension at a frequency of 40 kilohertz in a room temperature water bath for 10 minutes. Then, using an extruder, pass the sample through a membrane with a 400 nanometer pore size, 10 times.Follow this with a second cycle of extrusion, passing the sample through a membrane with a 200 nanometer pore size, four times. Set aside the resulting visibly clearer suspension, which contains large unilamellar vesicles of phospolipids about 175 nanometers in diameter. After the extrusion is complete, prepare standards in 13 by 100 millimeter glass tubes as outlined in the text protocol.Then, add 10 microliters of each LUV and proteoliposome sample to a unique glass tube and dilute with 40 microliters of deionized distilled water. Add 300 microliters of perchloric acid to each tube, and heat for one hour at 145 degrees Celsius in a heating block. Place a marble on each tube to prevent evaporation.Then let the tubes cool to room temperature and add one milliliter of deionized distilled water to each. Add 400 microliters of a freshly prepared solution containing 12 grams per liter of ammonium molybdate and 50 grams per liter of sodium ascorbate to each, and vortex to mix. Heat the tubes for 10 minutes at 100 degrees Celsius using marbles to prevent evaporation.Then remove the tubes from the heating block and allow them to cool to room temperature. Using a spectrometer set to 797 nanometers, obtain a calibration curve using the standards. Then, measure the absorbence of the samples against the blank, and determine the phosphate content.Begin by pipetting 800 microliters of each extruded LUV suspension into a unique two milliliter microfuse tube. Add 5.3 microliters of buffer A and 34.7 microliters of 10%mass-per-volume DDM, dissolved in buffer A, to each tube. Next, incubate the samples for three hours at room temperature with end-over-end mixing.While the samples are incubating, weigh out 400 milligrams per sample of beads in a glass beaker. Wash the beads with five milliliters per sample of methanol, stirring slowly for 10 minutes. Repeat this wash once for methanol, three times with water, and once with buffer A.During the last 30 minutes of sample incubation, dry 9.5 microliters-per-sample of NBD labeled phospholipid in a screw-cap glass tube under a stream of nitrogen.Then, add 45 microliters-per-sample of 0.1%mass-per-volume DDM in buffer A to dissolve the dried phospholipid. After incubation is complete, add the dissolved MBD labeled phospholipid solution to each sample. Then, add 0.1%DDM, buffer A, and the DDM-solubilized opsin in the desired amount, in that order, to obtain a final volume of one milliliter with a concentration of seven millimolar DDM.Reconstitution of a membrane protein is achieved by treating vesicles with sufficient detergent so that the swell will not dissolve. And that these conditions, which have to be empirically found for every lipid composition and detergent, the protein will integrate into the liposomes. Mix the samples for one hour at room temperature.Then, add 80 milligrams of the prepared polystyrene beads to each tube, and incubate again for one hour at room temperature with end-over-end mixing. Add an additional 160 milligrams of polystyrene beads to each sample, and incubate for two hours at room temperature with end-over-end mixing. When the incubation is complete, transfer the samples, leaving the beads behind, to clean, glass tubes, each containing 160 milligrams of fresh beads.Mix end-over-end overnight at four degrees Celsius, protected from light. Next, transfer the samples, without the beads, to unique microfuse tubes. Place the tubes on ice in preparation for the scramblase activity assay.Begin by adding 1950 microliters of buffer A to a plastic cubette, containing a plastic stir bar. Then, add 50 microliters of the prepared protealiposome sample. Let the sample equilibrate in the fluorescent spectrometer with constant stirring for five seconds.While the sample is equilibrating, prepare a solution of one molar dithionite in 0.5 molar unbuffered tris. Start the fluorescence monitoring with an excitation of 470 nanometers, an emission of 530 nanometers, and a slit width of 0.5 nanometers. Record for 50 seconds to achieve a stable signal.Then, add 40 microliters of one molar dithionite solution to the cubette. Record the fluorescence for at least 500 seconds. Finally, analyze the data as outlined in the text protocol.For a complete analysis of the fluorescence data, when it's to determine the fraction of vesicles that cannot be reconstituted. The recovery of proteins and lipids in the reconstituted vesicles and also the size distribution of the vesicles. In this study, opsin is reconstituted into large unilamellar vesicles to characterize its scramblase activity using a fluorescence-based assay.Vesicles are reconstituted with NBD-PC, which equally distributes between the outer and inner leaflets. Dithionite reduces the nitro group of NBD on the outside of the vesicle to a non-fluorescent amino groups resulting in a 50%reduction of fluorescence in protein-free liposomes. However, when opsin facilitates NBD movement between the inner and outer leaflet, a complete loss of fluorescence occurs.The representative results of this approach at different protein-to-phospholipid ratios can be seen here. The extend of fluorescence reduction increases with the amount of opsin reconstituted into vesicles, which ranges from zero to 4.95 micrograms. Dithionite was added at the indicated time, and NBD fluorescence was monitored for 400 seconds.Fluorescence reduction is more significant at higher protein-to-phospholipid ratios, with the largest reduction being 85%The endpoint fluorescence reduction is then used to determine the probability of scramblase activity, the likelihood of vesicles having at least one scramblase using Polson statistics. The red curve represents a mono-exponential fit for opsin. While the dashed grey lines represent mono-exponential fits for the reconstitution of opsin into vesicles as monomers, pre-formed dimers, or pre-formed tetramers.The method we describe can easily be adapted to different needs. The procedures are versatile, and can be used to identify and characterize phospholipid scramblase activity of other membrane proteins in the future. The reconstitution procedure can be applied to any membrane protein.This technique enabled us to identify opsin as the first biochemically verified phospholipid scramblase and allowed us, then, to characterize the activity of different confirmation states of the protein, and its ability to transport different lipids.

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Biochemistry

13.6K Aufrufe.  Weill Cornell Medical College. The overall goal of this procedure is to reconstitute a typical G protein coupled receptor into large unilamellar vesicles, and to measure it's ability to carry out 80p independent translocation of phospholipids across a lipid bilayer using a flurorescence-based assay. This method can help answer key questions in the inter-cellular lipid transport field, such as what is the molecular identity of proteins that can scramble off liplipids and what is their mechanism. The main advantage of this t...