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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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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Buffers used in the paper&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Buffer A</td><td></td><td></td><td>100 mM KCl, 50 mM MOPS pH 7.4, 1 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Buffer B</td><td></td><td></td><td>20 mM KCl, 10 mM MOPS pH 7.4, 0.1 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</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&lt;br/&gt; 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 ...

detergent-free-ultrafast-reconstitution-membrane-proteins-into-lipid

Research

JoVE Journal

Biochemistry

11.1K Views.  First Pavlov State Medical University. Hier presenteren we twee ultrasnelle protocollen voor reconstructie van membraaneiwitten in fusogenic proteoliposomes, en de fusie van dergelijke proteoliposomes met doel lipide bilayers voor wasmiddel-gratis bezorging van deze membraaneiwitten binnen de postfusion dubbelgelaagde. De combinatie van deze benaderingen kan snel en gemakkelijk-gecontroleerde vergadering van complexe multi-component membraan systemen.

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

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a &#8220;one-size-fits-all&#8221; approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one&#160;day in a detergent-free environment.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

Lipid Vesicle Encapsulation Protocol for Complex Protein Networks

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are&#160;trapped in microfluidic devices for analysis by optical microscopy.

Author Spotlight: Tackling Challenges in Synthetic Cell Engineering

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Neuroscience

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Biology

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

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

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