Name: detergent-free ultrafast reconstitution of membrane proteins into lipid bilayers using fusogenic complementary-charged proteoliposomes.
Uploaded: 2018-04-05T12:30:00
Duration: 11 min 10 s
Description: Watch this Scientific Journal Video about Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes. at JoVE.com

Authors are thankful to Robert Gennis from University of Illinois and Christoph von Ballmoos from University of Bern for providing a plasmid and a strain to express bo3-oxidase. The project was supported by BBSRC grant BB/L01985X/1 to R.I. and R.B.

<ol>
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G., 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=Determinants+of+liposome+fusion+mediated+by+synaptic+SNARE+proteins.">Determinants of liposome fusion mediated by synaptic SNARE proteins.</a> Proc Natl Acad Sci U S A. 101 (9), 2858-2863 (2004).</li><li>Spencer, A. C., Torre, P., Mansy, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+encapsulation+of+cell-free+transcription+and+translation+machinery+in+vesicles+for+the+construction+of+cellular+mimics.">The encapsulation of cell-free transcription and translation machinery in vesicles for the construction of cellular mimics.</a> J Vis Exp. 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H., Shorr, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Microcolorimetric+Method+for+the+Determination+of+Inorganic+Phosphorus.">A Microcolorimetric Method for the Determination of Inorganic Phosphorus.</a> Journal of Biological Chemistry. 202 (2), 675-685 (1953).</li><li>Galkin, M. A., Ishmukhametov, R. R., Vik, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functionally+inactive,+cold-stabilized+form+of+the+Escherichia+coli+F1Fo+ATP+synthase.">A functionally inactive, cold-stabilized form of the Escherichia coli F1Fo ATP synthase.</a> Biochimica Et Biophysica Acta-Bioenergetics. 1757 (3), 206-214 (2006).</li><li>Ishmukhametov, R. R., Galkin, M. A., Vik, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+purification+and+reconstitution+of+His-tagged+cysteine-less+Escherichia+coli+F1Fo+ATP+synthase.">Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1Fo ATP synthase.</a> Biochim Biophys Acta. 1706 (1-2), 110-116 (2005).</li><li>Wiedenmann, A., Dimroth, P., von Ballmoos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deltapsi+and+DeltapH+are+equivalent+driving+forces+for+proton+transport+through+isolated+F(0)+complexes+of+ATP+synthases.">Deltapsi and DeltapH are equivalent driving forces for proton transport through isolated F(0) complexes of ATP synthases.</a> Biochim Biophys Acta. 1777 (10), 1301-1310 (2008).</li><li>Morein, S., 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=Wild-type+Escherichia+coli+cells+regulate+the+membrane+lipid+composition+in+a+&amp;#34;window&amp;#34;+between+gel+and+non-lamellar+structures.">Wild-type Escherichia coli cells regulate the membrane lipid composition in a &amp;#34;window&amp;#34; between gel and non-lamellar structures.</a> J Biol Chem. 271 (12), 6801-6809 (1996).</li><li>Zinser, E., 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=Phospholipid+synthesis+and+lipid+composition+of+subcellular+membranes+in+the+unicellular+eukaryote+Saccharomyces+cerevisiae.">Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae.</a> J Bacteriol. 173 (6), 2026-2034 (1991).</li><li>Daum, G., Vance, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Import+of+lipids+into+mitochondria.">Import of lipids into mitochondria.</a> Prog Lipid Res. 36 (2-3), 103-130 (1997).</li><li>Pantazatos, D. P., MacDonald, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directly+observed+membrane+fusion+between+oppositely+charged+phospholipid+bilayers.">Directly observed membrane fusion between oppositely charged phospholipid bilayers.</a> J Membr Biol. 170 (1), 27-38 (1999).</li><li>Pantazatos, D. P., Pantazatos, S. P., MacDonald, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilayer+mixing,+fusion,+and+lysis+following+the+interaction+of+populations+of+cationic+and+anionic+phospholipid+bilayer+vesicles.">Bilayer mixing, fusion, and lysis following the interaction of populations of cationic and anionic phospholipid bilayer vesicles.</a> J Membr Biol. 194 (2), 129-139 (2003).</li><li>Sunami, T., 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=Detection+of+association+and+fusion+of+giant+vesicles+using+a+fluorescence-activated+cell+sorter.">Detection of association and fusion of giant vesicles using a fluorescence-activated cell sorter.</a> Langmuir. 26 (19), 15098-15103 (2010).</li><li>Memoli, A., 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=Effects+of+surfactants+on+the+spectral+behaviour+of+calcein+(II):+a+method+of+evaluation.">Effects of surfactants on the spectral behaviour of calcein (II): a method of evaluation.</a> J Pharm Biomed Anal. 19 (3-4), 627-632 (1999).</li></ol>

The authors have nothing to disclose.

The following few issues need to be considered for success of this experimental approach:
Choice of lipid charge for proteoliposomes and target bilayers: Cationic lipids are not found in nature, while anionic lipids are abundant in biological membranes reaching, for example, ~25, 35 and 20% in inner membrane of E. coli, plasma membrane of yeast S. cerevisiae, and inner mitochondrial membranes of many species, respectively27,28,29. It would be reasonable to expect that the functionality of membrane proteins in PL+ may be affected by the strength of a positive charge of the bilayer, which in turn would depend on a relative content of cationic lipid in the bilayer and external ionic strength. Therefore, it is important to address experimentally to what extent functionality of membrane proteins of interest would depend on the charge of the cationic lipid environment. Here, we show that both F1Fo ATP synthase and bo3-oxidase are sensitive to cationic lipid environment, but we managed to modulate and reverse this effect by first placing the proteins in PL+ and delivering them into anionic accepting bilayers, and then increasing the ionic strength of the reaction medium after fusion is finished.
Choice of a particular cationic lipid: Most commercially available cationic lipids are of non-triacylglyceride nature; therefore a potential candidate lipid must be tested for compatibility with membrane proteins of interest. We found previously15 that the ATP synthase used in this study showed its best performance in PL+ formed of ethyl-PC, which has the highest structural similarity to the natural triacylglyceride lipids, while in DOTAP (non-triacylglyceride lipid) PL+ this protein was less active.
Vesicle fusion assays: True vesicle fusion (when intravesicular liquid contents mix) needs to be differentiated from intermediate hemi-fusion states, when vesicles adhere to each other without mixing their aqueous content, but readily mix contents of their external or both lipid leaflets30. In case of suboptimal lipid mixtures or certain conditions, vesicles also demonstrate pronounced liquid content leakage during fusion31. Minimal concentrations of charged lipids in fusogenic mixtures enabling true fusion need to be found experimentally for each lipid species, but in general, it is found that membranes with less than 10% charged lipids are rendered non-fusogenic 32.
While forming fusogenic SUV in presence of multi-valent ions, it is important not to mix oppositely charged components, as this may cause immediate clumping and aggregation of lipids by these ions. For example, while preparing SUV for the cobalt-calcein-EDTA method, it is important to avoid mixing a cationic lipid mixture with EDTA to prevent clogging of the polycarbonate filter by clumped components.
It is also important to mention that the cobalt-calcein-EDTA method, while being very sensitive and convenient for real-time fusion monitoring, may still underestimate the extent of fusion due to 1) self-quenching of fluorescence of free calcein inside postfusion vesicles, which is expected to reach 1 mM, while the self-quenching threshold is reported to be around 20 &#181;M33, and 2) surface-bound cobalt-calcein, which remains bound to SUV+ even after passing through the gel-filtration resin and colors vesicles to a bright orange color, while SUV0 have a pale orange color. Also note that release of the bound cobalt-calcein upon addition of detergent Triton X-100 in the presence of EDTA generates much stronger signals for SUV+ (Figure 3C, red trace) than SUV0 (grey trace).
Perspectives: We expect that the fast approaches described here may greatly facilitate and speed up assembly of complex membranes for the needs of emerging synthetic biology applications. Our membrane protein reconstitution protocol takes only half an hour for reconstitution of fragile large membrane proteins known to be sensitive to lengthy dialysis-based reconstitution techniques, while this fusogenic approach takes only 5 - 10 min to deliver such proteins into large lipid bilayers. Here, we demonstrate the advantages of these approaches by manipulating E. coli F1Fo ATP synthase, which is an example of a fragile protein. It is made of 23 subunits and is known to readily lose its integrity if exposed to suboptimal conditions (for example, heat) during/after solubilization, but being used in these procedures, the protein demonstrates reproducibly high activity in proton pumping.

Functionalization of artificial lipid bilayers with membrane proteins is a key step in assembly of membrane model systems. The simplest model, proteoliposomes (PL), consists of small (30-200 nm diameter) unilamellar vesicles (SUV, also called liposomes), with proteins integrated into their membranes. PL are traditionally formed in two steps1. First, preformed SUV are mixed with a membrane protein of interest and a detergent at a concentration above its critical micelle concentration (CMC). Second, the detergent is removed with various dialysis, &#34;bio-beads&#34; or gel filtration techniques, leaving the protein in the membrane. The latter approach is much faster (~30 min1) and is therefore preferable for reconstitution of fragile and sensitive membrane proteins, while the first two approaches are limited by detergent removal speed, which takes many hours and may cause a substantial loss of activity and loss of structural integrity of the proteins. Functionalization of larger vesicles (large unilamellar vesicles, LUV, up to 1 &#181;m diameter) by this approach is more challenging, as vesicle size gets reduced after detergent removal, and it is not possible for giant unilamellar vesicles (GUV, &#62;1 &#181;m), as they are destabilized by detergents (but see Johnson et al.2 for slow 2D-crystallization of membrane proteins in large bilayers). Alternative approaches for GUV membrane functionalization3,4,5 exist but are laborious, time consuming, and still require some detergent at concentrations below CMC. More complex or fragile lipid models (for example, Droplet Hydrogel Bilayers6 and 3D printable Droplet Interface Bilayer-based artificial tissues7) cannot tolerate detergents. Quickly emerging synthetic biology applications8,9,10 critically depend on functionalization of such complex membrane structures. Therefore, an easy and robust method allowing fast and gentle delivery of membrane proteins into the target fragile bilayers is highly sought in the field.
An alternative, detergent-free protein delivery method is vesicle fusion, where interacting vesicles&#39; membranes unite into the intact postfusion bilayer, while their intravesicular aqueous contents get mixed, without being released into the external environment. Vesicle fusion is enabled and driven either by conformational rearrangements within complementary fusogenic agents (some proteins11,12 and peptides13 or specially modified DNA14) located in the contacting bilayers, or Coulombic interactions between lipid bilayers formed of complementarily charged cationic and anionic lipids15,16, or cationic bilayers and negatively charged proteins17.
The former approach requires the presence of fusogenic agents in the interacting membranes prior to fusion, is relatively slow (~30 min to reach half-maximum of fusion12,18), but can be applied to both natural and artificial membranes.
An advantage of the approach using fusogenic lipids (Figure 1) is that it enables much faster membrane fusion (~1 min to reach half-maximum, and 5-10 min to finish the reaction). Additionally, the extent of fusion can be controlled by i) an easy to formulate relative content of charged lipids in fusogenic bilayers, and ii) ionic and, in general, osmotic strength of the reaction medium (salts at above 50 mM and, for example, sucrose15 are shown to stop fusion), or a combination of both. To initiate fusion, oppositely charged fusogenic vesicles are mixed in a low (typically 10-20 mM salt) ionic strength medium for 5-10 min. A relative disadvantage of the method is that cationic lipids may exert a negative effect on functionality of membrane proteins in cationic proteoliposomes prior to fusion, especially in low ionic strength, but this effect is reversible and mitigated by a natural lipid composition of the post-fusion membrane and its return to the normal ionic strength medium.

<table>
	<tbody>
		<tr>
			<td>DOPC neutral lipid</td>
			<td>Avanti Lipids</td>
			<td>850375</td>
			<td></td>
		</tr>
		<tr>
			<td>POPA anionic lipid</td>
			<td>Avanti Lipids</td>
			<td>840857</td>
			<td></td>
		</tr>
		<tr>
			<td>E-PC cationic lipid</td>
			<td>Avanti Lipids</td>
			<td>890704</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesteryl-Bodipy-FL12</td>
			<td>Thermofisher</td>
			<td>C3927MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex G-50, Super Fine</td>
			<td>Sigma</td>
			<td>G5050</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400, Type 400-DL</td>
			<td>Sigma</td>
			<td>F8016</td>
			<td></td>
		</tr>
		<tr>
			<td>ACMA</td>
			<td>Sigma</td>
			<td>A5806</td>
			<td></td>
		</tr>
		<tr>
			<td>Nigericin</td>
			<td>Sigma</td>
			<td>N7143</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A26209</td>
			<td></td>
		</tr>
		<tr>
			<td>Q1</td>
			<td>Sigma</td>
			<td>C7956</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>PEP</td>
			<td>Sigma</td>
			<td>860077</td>
			<td></td>
		</tr>
		<tr>
			<td>NADH</td>
			<td>Sigma</td>
			<td>N8129</td>
			<td></td>
		</tr>
		<tr>
			<td>PK</td>
			<td>Sigma</td>
			<td>P9136-1KU</td>
			<td></td>
		</tr>
		<tr>
			<td>LDH</td>
			<td>Sigma</td>
			<td>L1254-1KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferin</td>
			<td>Sigma</td>
			<td>L6882</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferase</td>
			<td>Sigma/Roche</td>
			<td>10411523001</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Insight Biotechnology</td>
			<td>sc-202090</td>
			<td></td>
		</tr>
		<tr>
			<td>CoCl2</td>
			<td>Sigma</td>
			<td>C8661</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulforhodamine 101</td>
			<td>Sigma</td>
			<td>S7635</td>
			<td></td>
		</tr>
		<tr>
			<td>Extrusion system</td>
			<td>Avanti Extruder</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single tube luminometer, model Sirius L</td>
			<td>Titertek Berthold</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate filter, D19 mm</td>
			<td>Sigma, Whatman NucleporeTrack-Etched Membranes</td>
			<td>WHA800309, WHA800284</td>
			<td>100 or 200 nm to form SUV, and 400 or 800 nm for LUV</td>
		</tr>
		<tr>
			<td>Buffers used in the paper</td>
			<td>Composition</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Buffer A</td>
			<td></td>
			<td></td>
			<td>100 mM KCl, 50 mM MOPS pH 7.4, 1 mM MgCl2</td>
		</tr>
		<tr>
			<td>Buffer B</td>
			<td></td>
			<td></td>
			<td>20 mM KCl, 10 mM MOPS pH 7.4, 0.1 mM MgCl2</td>
		</tr>
		<tr>
			<td>Buffer C</td>
			<td></td>
			<td></td>
			<td>100 mM KCl, 10 mM MOPS pH 7.4</td>
		</tr>
		<tr>
			<td>Buffer D</td>
			<td></td>
			<td></td>
			<td>1 mM MOPS pH 7.4 
			Various concentrations of KCl</td>
		</tr>
	</tbody>
</table>

detergent-free ultrafast reconstitution of membrane proteins into lipid bilayers using fusogenic complementary-charged proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

The use of fusogenic complementary-charged proteoliposomes for fast detergent-free delivery of membrane proteins into target bilayer membranes includes three steps (Figure 1): A, formation of fusogenic SUV from lipid mixtures with high content of charged lipids; these SUV may optionally carry an intravesicular load; B, conversion of fusogenic SUV into PL using our fast membrane protein reconstitution; C, fusion of fusogenic PL with target bilayers in a low-salt medium, followed by addition of high salt to stop the fusion reaction. In case of large vesicles (D), a preferable strategy is to deliver membrane protein into the anionic target bilayer, which provides a better lipid environment for activity of membrane proteins (discussed further in the text).
The GUV formation protocol with inverted emulsion method is highlighted in detail in Figure 2. We prefer to use hexadecane in the lipid-oil mixture due to its relatively high (18 &#176;C) freezing temperature, which allows easy removal of solidified oil after GUV pelleting.
Figure 3 demonstrates vesicle fusion using cobalt-calcein-EDTA method. Fusion is seen only when complementary charged vesicles are used in low salt buffers, while higher salt concentrations (&#62;50 mM14) or the use of non-fusogenic vesicles demonstrate no fusion.
This fast protocol of reconstitution of membrane proteins into fusogenic SUV is illustrated in Figure 4A. Using this protocol, we demonstrate fast reconstitution of primary proton pump bo3-oxidase and F1Fo ATP synthase into PL, and assessment of their specific activity in these membranes. It is important to mention that the yield of protein reconstitution does not depend on the charge of a lipid used15 and is about 50 - 75%25,12, and that after reconstitution into PL, the protein can be stored for at least three days, even at room temperature without obvious loss of protein activity. This procedure also provides unidirectional orientation of ATP synthase, where more than 95% of it has its hydrophilic moiety F1 oriented outwards15,26.
Figure 5 demonstrates that the proton pumping activity of F1Fo (Panel A) and bo3-oxidase (Panel B) in cationic lipids is reduced and sensitive to low ionic strength, as compared to anionic and neutral lipid environment, but this effect is mitigated in postfusion membranes after fusing PL+ with anionic LUV-.
Figure 6 demonstrates delivery of F1Fo ATP synthase into membranes of large vesicles. In this experiment, PL+ and 800 nm LUV- were fused in 20 mM KCl for 5 min, and the reaction product was pelleted to remove unfused PL+, resuspended and assayed for proton pumping. Control experiments showed no proton pumping when LUV0 instead of LUV- were used in fusion reaction (black trace), or empty LUV- alone were assayed (blue trace).
Fast assembly of a functioning electron transport chain in membranes of large vesicles by means of fusogenic PL is shown in Figure 7. We used F1Fo SUV+ and bo3 SUV+ for fusion with 800 nm LUV-, and demonstrated ATP production by this chain in the postfusion vesicles by sequentially adding Coenzyme Q1 and DTT to energize membranes, and then adding phosphate to trigger ATP synthesis by F1Fo.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56909/56909fig1.jpg" /> 
Figure 1: Conception of ultrafast detergent-free delivery of membrane proteins into target lipid bilayers via fusion of unilamellar vesicles formed of complementary charged lipids. (A) formation of cationic and anionic fusogenic small unilamellar vesicles (SUV+, SUV-) optionally loaded with intravesicular cargoes. (B) Conversion of fusogenic SUV into fusogenic proteoliposomes (PL) by reconstitution of membrane proteins. (C) Detergent-free delivery of membrane proteins into postfusion membranes with fusogenic PL. (D) Preferable strategy for delivery of membrane proteins into membranes of large vesicles, illustrated by the fusion between 100 nm PL+ and 800 nm LUV-. <a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56909/56909fig2.jpg" /> 
Figure 2: GUV formation protocol with an inverted emulsion method. (A) Formation of a lipid monolayer on the border between the oil-lipid mixture and water. (B) Formation of an inverted (water-in-oil) emulsion. (C) GUV formation by passing the emulsion through the oil-water border by means of centrifugation. (D) Cooling of the tube below &#60;18 &#176;C to solidify and remove the oil. (E) A fluorescent microscopy image of a GUV containing fluorescent lipid (1% weight fraction cholesteryl-Bodipy-FL12) in its membrane (green) and a polar fluorophore (1 mM Sulforhodamine 101) in vesicle`s lumen (red). <a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56909/56909fig3.jpg" /> 
Figure 3: Lipid vesicles fusion studied with the cobalt-calcein-EDTA method. (A) Schematic of the method, where free calcein is released from a non-fluorescent cobalt-calcein complex by EDTA. (B) Fusion of SUV in various KCl concentrations. (C) Release of the intravesicular calcein by addition of EDTA and Triton X-100 detergent to postfusion vesicles shown in B as described in text. The fusion extent (%) for red trace is calculated by dividing the maximal background-corrected fusion signal (1 - 2) by the background-corrected maximal signal following the release of encapsulated calcein (3 - 2) and multiplying by 100. <a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56909/56909fig4.jpg" /> 
Figure 4: Ultrafast reconstitution of bo3-oxidase and F1Fo into fusogenic proteoliposomes, and protein activity measurements in such proteoliposomes. (A) Schematic of the reconstitution protocol. (B) Coenzyme Q1 oxidation driven proton pumping by bo3-oxidase in PL measured with ACMA quenching (explained in text). (C) ATP hydrolysis-driven proton pumping by F1Fo in PL measured with ACMA quenching (explained in text). (D) ATP hydrolysis by F1Fo in PL measured with ATP-regenerating system (explained in text), and its stimulation by the uncoupler. <a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56909/56909fig5.jpg" /> 
Figure 5: Influence of the lipid environment and ionic strength on activity of membrane proteins. Proton pumping by F1Fo (A) and bo3-oxidase (B) in cationic PL (red trace), anionic PL (blue trace), neutral PL (black trace), and cationic PL fused with anionic LUV (green trace) in 20 and 100 mM KCl. Control trace (gray) shows no change in proton pumping by F1Fo PL- upon mixing with SUV-. <a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56909/56909fig6.jpg" /> 
Figure 6: Delivery of F1Fo ATP synthase into membranes of 800 nm LUV- via fusion with PL+. (A) schematic of the experiment: PL+ and 800 nm LUV- were fused in 1 mM MOPS (pH 7.4), 1 mM MgCl2, 20 mM KCl for 5 min, pelleted to remove unfused PL, and resuspended in the same buffer. (B) Proton pumping by the postfusion LUV (red trace). Control experiments showed no ACMA quenching when PL0 were mixed with LUV- (black trace), or empty LUV- alone were assayed (blue trace). <a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56909/56909fig7.jpg" /> 
Figure 7: 5-min detergent-free assembly of an electron transport chain in membranes of 800 nm LUV- via fusion with PL+. (A) schematic of the experiment: 100 nm F1Fo PL+ and 100 nm bo3-oxidase PL+ were fused with LUV-, as described in Figure 6. Fusion was stopped by adding KCl and MOPS to 100 and 50 mM, respectively. The membranes were mixed with ADP-luciferin-luciferase cocktail and energized by addition of DTT and Q1, as described in the text. ATP production was initiated by addition of 1 mM phosphate (Pi) and monitored real time with luciferase-luciferin system as described in the text. (B) ATP synthesis by postfusion vesicles (red trace). Control experiments showed no ATP production when PL+ were mixed with LUV- in high salt (grey), or PL0 were mixed with LUV- (black). ATP synthesis rate was calculated as explained in the text (steps 8.8 - 8.9). <a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes. at JoVE.com

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

The overall goal of our easy to implement procedure is to demonstrate fast detergent-free delivery of membrane proteins into lipid bilayers of various sizes using fusion between lipid bilayers formed of oppositely charged lipids. Our methodology can be used in biochemistry and biophysics for one step assembly of biomer metric multi-component lipid bilayer systems from layer individual components. It is important to mention that the advantage of our approach is in this fast functionalizition, or namely complex model lipid bilase, with proteins, lipids and other molecules of interest.This methodology can also be used to study membrane transport processes that require energization of the lipid membrane. For example, primary pumps can be used to drive secondary transport enabled by other membrane proteins. Our method can also serve in needs of synthetic biology.For example, to create membrane vesicles with various program functionalities. In future, trapping small vesicles like these inside similar larger ones, maybe a step toward construction of an artificial cell. To form the lipid and oil solution, mix Chloroform stock with one milliliter of hexadecane in a 1.5 milliliter micro centrifuge tube.Next, to evaporate the Chloroform, constantly mix and heat the mixture in a horizontal shaker at 80 degrees Celsius for 30 minutes by leaving the tube open. After evaporation, close the tube and cool down to room temperature. Next, to form the lipid monolayer, at the lipid in oil aqueous buffer interface, take a 1.5 milliliter centrifuge tube, and transfer 200 microliters of lipid in oil solution on top of 0.5 milliliters of Buffer B.Then wait for 30-60 minutes at room temperature for the phase separation border to flatten, indicating the formation of the lipid monolayer.The convex shape of the phase separation border is due to surface tension mismatch between the oil and the aqueous buffer. Next, to prepare the water and oil emulsion, mix a solution of non-ionic poly saccharide in Buffer B with a density high than that of water. Then transfer 0.5 microliters of the mixture to a new 1.5 milliliter centrifuge tube, containing 100 microliters of lipid in oil solution.Then sonicate the mixture at 44 kilohertz and 14 watts for 30 seconds in an ultra sonic water bath. After sonicating, vigorously vortex for 45 minutes to form the water in oil emulsion. Then, to convert the water in oil emulsion into anionic GUV carefully transfer the emulsion on top of lipid and oil aqueous interface.In the emulsion, each droplet is coated with lipid monolayer. Proper syndication vortexing of the water in oil emulsion determines success of GUV formation. If not formed correctly, the emulsion droplet will coalesce in turn, reducing the yield of GUV formation.To obtain the GUV pellet, quickly centrifuge the sample at 1000 times G for two minutes. Place a bent metal wire in the tube so that it sits within the oil. Then leave the tube on ice to solidify the lipid and oil mixture.Carefully remove the frozen oil and collect the aqueous phase. Next, re-suspend the GUV pellet in 50 microliters of fresh Buffer B and transfer to a new tube. Inspect the GUV under a 100 X oil emersion objective of a fluorescent microscope.Use gel filtration resin to prepare a gravity flow column and equivalate with Buffer A kept at room temperature. Then dilute the purified membrane protein to a final concentration of 0.7 milligrams per milliliter using the extraction buffer. Then, mix the protein with pre-formed cationic SUV in a one to 30 ratio along with collate containing Buffer A to a final volume of 660 microliters.Then, place the sample tube on a rocker and gently shake the mixture for 15 minutes. Next, pass the mixture through gel filtration resin and collect the turbid proteoliposome fractions and centrifuge at 4, 000 times G for 15 minutes. Then, discard the non-reconstituted protein supernatant and dissolve the pellet in one milliliter of fresh Buffer A.In a fluorometer cuvette, add 20 microliters of proteoliposome with B03 oxidase along with two milliliters of Buffer A in the presence of 0.5 micromolar ACMA, and wait for stable signal at 430 nanometers excitation, and 515 nanometers emission.Then, add 40 micromolar of coenzyme Q1 to the cuvette. Next, add two millimolar DTT to the proteoliposome to start proton pumping, and then dissipate the formed delta pH. In a separate cuvette, add 40 microliters of proteoliposomes with F1-F0 to 2 milliliters of Buffer A in the presence of 0.5 micromolar ACMA and trigger proton pumping by adding 0.2 millimolar ATP to the proteoliposome and then dissipate the formed delta pH.Fuse 50 microliters of PL+with 50 microliters of 800 nanometers LUV-in one milliliter of Buffer D, supplemented with one millimolar of Magnesium Chloride, and 20 millimolar Potassium Chloride for five minutes. Then centrifuge the sample at 6, 000 times G for five minutes to pellet the post fusion LUV. After centrifugation, discard the supernatant consisting of unreacted PL+then resuspend the pellet formed in one milliliter of Buffer D, supplemented with one millimolar of Magnesium Chloride and 100 millimolar Potassium Chloride.Again, conduct the ACM Quenching Assay. First, mix together three microliters of B03 oxidase PL+and five microliters of F1F0 PL+then fuse the mixture with three microliters of LUV-or GUV-in 800 microliters in Buffer D, supplemented with 20 millimolar Potassium Chloride and one millimolar Magnesium Chloride for five minutes. Next, add Potassium Chloride and Mops Buffer to the fused membrane mixture at a final concentration of 150 millimolar.Mix 400 micromolar ADP, 50 micromolar luciferin and 2.5 of luciferase in 200 microliters of Buffer A to prepare a luciferin luciferase ADP cocktail. Then, add the cocktail to the fused membrane mixture. For correct ADP synthesis measurement, it is important to have a stable luciferase signal to avoid false positive, before addition of Q1 and DTT.Then add 40 micromolar oxidized coenzyme Q1, and then add two millimolar DTT to initiate the energization of the fused membrane. After one minute, add a five millimolar Potassium Phosphate solution to the energized vesicles to start ATP synthesis. To inspect the resuspended GUV on removing the frozen hexadecane from the lipid and oil mixture, fluorescent microscopy was utilized.Images of the GUV obtained show the presence of fluorescent cholesterol biopoty FL12 on its membrane, and self erodimine 101, a polar fluorophore in the lumen. To quantify the fusion of SUV in the increasing concentration of Potassium Chloride, a graph was plotted. The graph explains the with increasing Potassium Chloride concentration beyond 50 millimolar, the fusion of the SUV lipid vesicles starts decreasing.However, at low Potassium Chloride concentration, between one to 20 millimolar, the complementary charged vesicles fuse. Next, to measure the BO3 oxidase meditated proton pumping drive oxidation of coenzyme Q1 substrate, a graph was plotted based on ACMA Quenching Assay. The results show that adding an uncoupler increase the ACMA Fluorescence signal represented along the Y axis of the plot.Then, ATP hydrolysis dependent proton pumping by F1F0 was also measured based on ACMA Quenching Assay. In the graph, it shows that adding an uncoupler increases the AMCA Fluorescence signal. Further, luciferin luciferase system was used to monitor ATP synthesis in fused vesicles.Sequential addition of coenzyme Q1 DTT, followed by Phosphate addition, shows successful ATP production by the fused vesicles. Once mastered, the first technique formation of fusogentic proteoliposome from detergents symbolized membrane proteins and union myomedicals can be done in just 30 minutes. The second technique, physical fusion, can be done even faster within just ten minutes.After watching this video, you should have a good understanding of how one could construct multi-component membrane systems by combining various individual building blocks that are presented by functionalized lipid vesicles. We expect such modular systems where the chief properties and functions that are impossible with one step re-constitution of membrane proteins. In general, our procedures provide a simple framework for building complex compartmentalized bio-chemical systems, from simpler, so-called bio-bricks.

Research

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