Name: detergent-assisted reconstitution of recombinant drosophila atlastin into liposomes for lipid-mixing assays
Uploaded: 2019-07-03T13:00:00
Description: Watch this Scientific Journal Video about Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays at JoVE.com

We thank Dr. Michael Stern and his lab for their insights and feedback on atlastin related projects. This work was supported by the National Institute of General Medical Sciences [R01GM101377] and the National Institute of Neurological Disorders and Stroke [R01NS102676].

1. Purification of GST-DAtl-His8
<ol>
	<li>Protein expression and lysate preparation
	<ol>
		<li>Transform BL21 (DE3) E. coli with the GST-DAtl-His8 construct in pGEX4-T32 and select on an ampicillin plate.</li>
		<li>Select a single transformant and inoculate 5 mL of LB + ampicillin (5 &#181;L of 100 mg/mL ampicillin) in a 14 mL culture tube and incubate at 25 &#176;C with shaking at 200 rpm for 6-8 h. 
		NOTE: Due to leaky expression, it is not recommended to...

<ol>
	<li>Chernomordik, L. V., Kozlov, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+membrane+fusion.">Mechanics of membrane fusion.</a> Nature Structural and Molecular Biology. 15, 675-683 (2008).</li><li>Orso, 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=Homotypic+fusion+of+ER+membranes+requires+the+dynamin-like+GTPase+Atlastin.">Homotypic fusion of ER membranes requires the dynamin-like GTPase Atlastin.</a> Nature. 460, 978-983 (2009).</li><li>McNew, J. A., Sondermann, H., Lee, T., Stern, M., Brandizzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTP-dependent+membrane+fusion.">GTP-dependent membrane fusion.</a> Annual Review of Cell and Developmental Biology. 29, 529-550 (2013).</li><li>Rigaud, J. L., Pitard, B., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+into+liposomes:+application+to+energy-transducing+membrane+proteins.">Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1231, 223-246 (1995).</li><li>D&#252;zg&#252;ne&#351;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Assays+for+Liposome+Fusion.">Fluorescence Assays for Liposome Fusion.</a> Methods in Enzymology. 372, 260-274 (2003).</li><li>Wilschut, J., Duzgunes, N., Fraley, R., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mechanism+of+membrane+fusion:+kinetics+of+calcium+ion+induced+fusion+of+phosphatidylserine+vesicles+followed+by+a+new+assay+for+mixing+of+aqueous+vesicle+contents.">Studies on the mechanism of membrane fusion: kinetics of calcium ion induced fusion of phosphatidylserine vesicles followed by a new assay for mixing of aqueous vesicle contents.</a> Biochemistry. 19, 6011-6021 (1980).</li><li>Ellens, H., Bentz, J., Szoka, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton-+and+calcium-induced+fusion+and+destabilization+of+liposomes.">Proton- and calcium-induced fusion and destabilization of liposomes.</a> Biochemistry. 24, 3099-3106 (1985).</li><li>Meers, P., Ali, S., Erukulla, R., Janoff, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+inner+monolayer+fusion+assays+reveal+differential+monolayer+mixing+associated+with+cation-dependent+membrane+fusion.">Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1467, 277-243 (2000).</li><li>Schaffner, W., Weissmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+sensitive,+and+specific+method+for+the+determination+of+protein+in+dilute+solution.">A rapid, sensitive, and specific method for the determination of protein in dilute solution.</a> Analytical Biochemistry. 56, 502-514 (1973).</li><li>MacDonald, R. C., 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=Small-volume+extrusion+apparatus+for+preparation+of+large,+unilamellar+vesicles.">Small-volume extrusion apparatus for preparation of large, unilamellar vesicles.</a> Biochimica Et Biophysica Acta. 1061, 297-303 (1991).</li><li>Paternostre, M. T., Roux, M., Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+membrane+protein+insertion+into+liposomes+during+reconstitution+procedures+involving+the+use+of+detergents.+1.+Solubilization+of+large+unilamellar+liposomes+(prepared+by+reverse-phase+evaporation)+by+Triton+X-100,+octyl+glucoside,+and+sodium+cholate.">Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (prepared by reverse-phase evaporation) by Triton X-100, octyl glucoside, and sodium cholate.</a> Biochemistry. 27, 2668-2677 (1988).</li><li>Scott, B. L., 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=Liposome+Fusion+Assay+to+Monitor+Intracellular+Membrane+Fusion+Machines.">Liposome Fusion Assay to Monitor Intracellular Membrane Fusion Machines.</a> Methods in Enzymology. 372, 274-300 (2003).</li><li>Zhang, W., 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=Crystal+structure+of+an+orthomyxovirus+matrix+protein+reveals+mechanisms+for+self-polymerization+and+membrane+association.">Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.</a> PNAS. 114, 8550-8555 (2017).</li><li>Parlati, F., 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=Rapid+and+efficient+fusion+of+phospholipid+vesicles+by+the+&#945;-helical+core+of+a+SNARE+complex+in+the+absence+of+an+N-terminal+regulatory+domain.">Rapid and efficient fusion of phospholipid vesicles by the &#945;-helical core of a SNARE complex in the absence of an N-terminal regulatory domain.</a> PNAS. 96, 12565-12570 (1999).</li><li>Sugiura, S., Mima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+lipid+composition+is+vital+for+homotypic+ER+membrane+fusion+mediated+by+the+dynamin-related+GTPase+Sey1p.">Physiological lipid composition is vital for homotypic ER membrane fusion mediated by the dynamin-related GTPase Sey1p.</a> Scientific Reports. 6, 20407 (2016).</li><li>Powers, R. E., Wang, S., Liu, T. Y., Rapoport, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+tubular+endoplasmic+reticulum+network+with+purified+components.">Reconstitution of the tubular endoplasmic reticulum network with purified components.</a> Nature. 543, 257-260 (2017).</li><li>Betancourt-Solis, M. A., Desai, T., McNew, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+atlastin+membrane+anchor+forms+an+intramembrane+hairpin+that+does+not+span+the+phospholipid+bilayer.">The atlastin membrane anchor forms an intramembrane hairpin that does not span the phospholipid bilayer.</a> Journal of Biological Chemistry. 293, 18514-18524 (2018).</li></ol>

The methods here delineate an efficient method for purifying, reconstituting, and measuring fusion activity of recombinant atlastin. To ensure high yields of functional atlastin some critical steps must be considered. Expression of atlastin must be done at low temperatures (16 &#176;C) to avoid aggregation and one should aim for a final concentration between 0.4&#8211;1.5 mg/mL. Very dilute protein will not be reconstituted optimally at a 1:400 protein to lipid ratio. Reconstitution efficiency can be optionally analyzed ...

Membrane fusion is a critical process in many biological reactions. Under biological conditions, membrane fusion is not spontaneous and requires specialized fusion proteins to catalyze such reactions1. ER homotypic membrane fusion is mediated in animals by the dynamin related GTPase atlastin2. Atlastin&#8217;s role in homotypic fusion is fundamental for three-way junctions in peripheral ER, which constitutes a large interconnected network of tubules that extend throughout the cell. Atlastins have a conserved domain morphology consisting of a large GTPase, a three helix bundle middle domain, a hydrophobic membrane anchor, and a short cytoplasmic C-terminal tail3. In vitro studies with recombinant Drosophila atlastin have shown that when reconstituted to liposomes, it maintains its fusogenic properties. Other atlastins, including human homologs have not been able to recapitulate fusion in vitro. We describe here a methodology for purifying a GST and poly-histidine tagged recombinant Drosophila atlastin, reconstituting it to liposomes, and assaying fusion.
Studying membrane fusion in vitro presents a challenge as fusogenic proteins usually have a membrane anchor. In order to study them, it is necessary to reconstitute them into model lipid bilayers. Large unilamellar vesicles (LUV) are a useful tool to study lipid protein interactions. We present here a system to make LUVs of different lipid compositions for protein reconstitution and fusion assays. Reconstitution of integral proteins into LUVs can be achieved by a variety of methods including, organic solvent-mediated reconstitution, mechanical mechanisms, or detergent assisted reconstitution4. We present here a method for reconstituting Drosophila atlastin into preformed liposomes by detergent removal. Advantages of this reconstitution method include high reconstitution yields and proper orientation of atlastin in the lipid bilayer. Additionally, through this method, the protein is not dried or exposed to organic solvents thereby maintaining structure and function. Among its disadvantages, the presence of detergents may not be ideal for all proteins and the final proteoliposomes may have some incorporated detergent in the lipid bilayer. Further dialysis may be used to eliminate more of the detergent. However, dialysis may take a long time and can therefore lead to loss of protein activity.
Assessing atlastin&#8217;s fusion activity can be determined by lipid mixing assays as previously described2. Here, we delineate a method for measuring atlastin mediated fusion through N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)/Lissamine rhodamine-B sulfonyl (rhodamine) labeled lipids. This assay requires fusion of donor (labeled) proteoliposomes and acceptor (unlabeled) proteoliposomes. A FRET release can be measured during the reaction as the dilution&#160;of a donor&#8211;acceptor pair from &#8220;labeled&#8221; liposomes to &#8220;unlabeled&#8221; liposomes as a result of lipid mixing during membrane fusion (Figure 1)5. While this assay serves as a proxy for membrane fusion, it is limited in distinguishing between membrane fusion and hemifusion, a state where only the outer leaflets mix. To address this issue, an alternative is outer leaflet quenching of NBD by dithionite. Following the same methodology as NBD/rhodamine lipid mixing assays, upon quenching the outer leaflet any NBD FRET release by fusion will be due to inner leaflet mixing8.
Alternative fusion assays by inner aqueous content mixing address full fusion only5. Examples of this are terbium (Tb)/dipicolinic acid (DPA) assays and aminonaphthalene trisulfonic acid (ANTS)/p-xylene bis(pyridinium) bromide (DPX) assays. In Tb/DPA assays, a pool of liposomes with encapsulated Tb are mixed and fused with liposomes with encapsulated DPA; upon fusion, fluorescence is increased via internal energy transfer from DPA to Tb within the [Tb(DPA)3]3- chelation complex6. In contrast, for ANTS/DPX assays, ANTS fluorescence is quenched by DPX7. While these systems address inner content mixing, more in-depth preparation of liposomes is required for removal of non-encapsulated reagents, as well as unintended interaction of the fluorophores.

<table>
	<tbody>
		<tr>
			<td>10 mL poly-prep chromatography columns</td>
			<td>Biored</td>
			<td>731-1550</td>
			<td></td>
		</tr>
		<tr>
			<td>10 x 75 mm Flint glass tubes</td>
			<td>VWR</td>
			<td>608225-402</td>
			<td></td>
		</tr>
		<tr>
			<td>47 mm diameter, 0.45um pore whatman sterile membrane filters</td>
			<td>Whatman</td>
			<td>7141 104</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well white plate</td>
			<td>NUNC</td>
			<td>437796</td>
			<td></td>
		</tr>
		<tr>
			<td>Anapoe X-100</td>
			<td>Anatrace</td>
			<td>9002-931-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell disrupter</td>
			<td>Avestin</td>
			<td>Avestin Emulsiflex C3</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt))</td>
			<td>Avanti</td>
			<td>840035P-10mg</td>
			<td>DOPS</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Research organics inc.</td>
			<td>6381-92-6</td>
			<td>Ethylenediaminetetraacetic acid</td>
		</tr>
		<tr>
			<td>EDTA-free protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td>Complete protease inhibitor</td>
		</tr>
		<tr>
			<td>Extruder</td>
			<td>Sigma Aldrich</td>
			<td>Z373400&#160;</td>
			<td>Liposofast Basic Extruder</td>
		</tr>
		<tr>
			<td>GE Akta Prime liquid chromatography system</td>
			<td>GE Pharmacia</td>
			<td>8149-30-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione agarose beads</td>
			<td>Sigma aldrich</td>
			<td>G4510-50ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>EMD</td>
			<td>GX0185-5</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Sigma Aldrich</td>
			<td>36051-31-7</td>
			<td>Guanosine 5&#39; triphosphate sodium salt hydrate</td>
		</tr>
		<tr>
			<td>HEPES, acid free</td>
			<td>Omnipur</td>
			<td>5330</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>fluka</td>
			<td>5670</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilized metal affinity chromatography (IMAC) resin column</td>
			<td>GE Healthcare</td>
			<td>17040801</td>
			<td>1 mL HiTrap Chelating HP immobilized metal affinity chromatography columns</td>
		</tr>
		<tr>
			<td>Iohexol</td>
			<td>Accurate chemical and scientific corporation</td>
			<td>AN 7050 BLK</td>
			<td>Accudenz/Nycodenz</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Research products international corp.</td>
			<td>I56000-100.0</td>
			<td>IPTG, dioxane free</td>
		</tr>
		<tr>
			<td>L-Glutathione reduced</td>
			<td>Sigma-Aldrich</td>
			<td>G4251-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Omnisolv</td>
			<td>MX0488-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NBD-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt))</td>
			<td>Avanti</td>
			<td>236495</td>
			<td>NBD-DPPE</td>
		</tr>
		<tr>
			<td>n-Dodecyl &#946;-D-maltoside</td>
			<td>Chem-Impex International</td>
			<td>21950</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonpolar polystyrene adsorbent beads</td>
			<td>BioRad</td>
			<td>152-3920</td>
			<td>SM2 Biobeads</td>
		</tr>
		<tr>
			<td>Nuclepore track-etch polycarbonate 19 mm 0.1 um pore membrane</td>
			<td>Whatman</td>
			<td>800309</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima LE80K Ultra centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylcholine, L-&#945;-dipalmitoyl [choline methyl-3H]</td>
			<td>ARC</td>
			<td>ART0284</td>
			<td>Titriated lipids</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>TECAN</td>
			<td>TECAN infinite M200 plate reader</td>
			<td></td>
		</tr>
		<tr>
			<td>POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine)</td>
			<td>Avanti</td>
			<td>850457C-25mg</td>
			<td>POPC</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>MP</td>
			<td>151944</td>
			<td></td>
		</tr>
		<tr>
			<td>Rh-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt))</td>
			<td>Avanti</td>
			<td>236495</td>
			<td>Rh-DPPE</td>
		</tr>
		<tr>
			<td>Scintillation Cocktail</td>
			<td>National Diagnostics</td>
			<td>LS-272</td>
			<td>Ecoscint XR Scintillation solution for aqueous or non-aqueous samples</td>
		</tr>
		<tr>
			<td>Scintillation vials</td>
			<td>Beckman</td>
			<td>592928</td>
			<td>Fast turn cap Mini Poly-Q Vial</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T1063-1kU</td>
			<td>Thrombin from human plasma</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear centrifuge tubes 5 x 41 mm</td>
			<td>Beckman</td>
			<td>344090</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex 9 to 13mm Tube Holder</td>
			<td>VWR</td>
			<td>58816-138</td>
			<td>Insert for vortexing flint glass tubes</td>
		</tr>
		<tr>
			<td>Vortex Insert Retainer</td>
			<td>VWR</td>
			<td>58816-132</td>
			<td>Retainer needed for vortex tube holder</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>VWR</td>
			<td>2.235074</td>
			<td>Vortex Genie 2 model G560</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol molecular biology grade</td>
			<td>Calbiochem</td>
			<td>444203</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The efficiency of atlastin reconstitution is presented in Figure 2. Reconstituted proteoliposomes were floated in an iohexol discontinuous gradient. Unincorporated protein was sedimented in the bottom layer (B) or in the middle layer (M). Reconstituted protein would float to the top layer (T). Samples of the gradient were harvested and analyzed by SDS-PAGE and Coomassie staining. The quantification of the gel by densitometry shows a very high efficiency of reconstitution with negligible lose...

Watch this Scientific Journal Video about Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays at JoVE.com

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.

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 ...

The reconstitution and fusion protocol detailed here is an efficient way to study membrane-bound proteins in a model lipid bilayer and lipid mixing by fusion proteins. Here we describe a detergent-assisted reconstitution of recombinant Drosophila atlastin, an ER fusion protein, into preformed phosphatidylcholine liposomes. Fusion of atlastin proteoliposomes is then measured by a FRET-based lipid-mixing assay.One of the key advantages of detergent-assisted reconstitution is that the protein in detergent is not exposed to drying or solvents. We also show here that reconstitution orientation of atlastin is very efficient. Another advantage of the FRET-based lipid-mixing assay is that the liposomes do not need to be loaded with any dye, and there's no need for any extra dialyses steps.To begin this procedure, prepare the lipid mix stocks in chloroform, as outlined in the text protocol. Add one microcurie per milliliter of DPPC to the lipid mixes, making sure to reserve at least eight microliters of this stock. Transfer the desired amount of the lipid mixes into flint glass tubes.Then dry the lipid mixes under a gentle stream of nitrogen gas for approximately 10 minutes until no more chloroform is visible. Dry the resulting lipid film further in a desiccator by vacuuming for 30 minutes. After this, add enough A100 with 10%glycerol, two-millimolar 2-mercaptoethanol, and one-millimolar EDTA to the lipid film to bring the concentration back to 10 millimolar.Resuspend the lipid film by vortexing lightly for 15 minutes at room temperature. Freeze the hydrated lipids in liquid nitrogen. To thaw the liposomes, let the solution sit at room temperature for 30 seconds, then transfer them back to water for faster thawing.Repeat this freeze-thaw cycle a total of 10 times. After this, use a mini-extruder to pass the lipids through polycarbonate filters, with a pore size of 100 nanometers, 19 times. To determine the total lipid concentration of the liposomes, add four microliters of lipid stock and lysosomes to three milliliters of scintillation cocktail.Measure the average counts per minute for stock and liposomes, and calculate the liposome concentration, as outlined in the text protocol. Store the liposomes at four degrees Celsius for up to one week. First, mix the appropriate amount of buffer, extra detergent, and protein in a 0.5-milliliter tube, as outlined in the text protocol.Rapidly add the liposomes, and immediately vortex for five seconds to homogenize the mixture. Incubate the reaction in a nutator at four degrees Celsius for one hour. During this incubation, weigh out 0.2 grams of polystyrene adsorbent beads and transfer them to a microcentrifuge tube.Add one milliliter of methanol to the tube, and mix for one minute to degas the beads. Then, aspirate the methanol with a 21-gauge or higher needle. To begin washing the beads, add water to them.Let the beads mix with the water for five minutes, then aspirate the water. Repeat this water washing process four times. After this, add water to bring the polystyrene adsorbent bead slurry to a volume of one milliliter, with a final concentration of 0.2 grams per milliliter.Calculate the amount of polystyrene adsorbent beads needed to adsorb all the detergent in each sample, as outlined in the text protocol. Cut a pipette tip to collect the bead slurry. Transfer the calculated amount of bead slurry to a 0.5-milliliter tube, and aspirate the water.Add the samples to the tube containing the beads, and incubate in a nutator at four degrees Celsius for one hour. Transfer the samples to a fresh tube containing new beads, making sure to leave the old beads behind. Incubate again using the same conditions.Repeat this process of transferring and incubating the beads two times. After this, transfer the sample to a new tube containing fresh beads and incubate by nutating overnight at four degrees Celsius. The next day, remove the sample from the beads and centrifuge at 16, 000 times g and four degrees Celsius for 10 minutes to pellet the insoluble protein aggregates.Recover the supernatant, and determine the final lipid concentration by liquid scintillation counting, as outlined in the text protocol. To begin, bring the donor and acceptor proteoliposomes to a concentration of 0.15 millimolar each in A100 containing glycerol, beta-mercaptoethanol, EDTA, and magnesium chloride. Transfer each reaction to a well in a flat 96-well plate suitable for fluorescence reading, making sure to prepare at least four reactions, including a triplicate and a no-GTP negative control.Place the plate into a preheated plate reader at 37 degrees Celsius. Measure the NBD fluorescence every minute for five minutes. Then, add five-millimolar GTP to induce fusion.Measure the NBD fluorescence every minute for one hour using the same excitation and emission as before. After this, add five microliters of 2.5%n-Dodecyl beta-D-maltoside to the wells to dissolve the proteoliposomes, and measure the maximum NBD fluorescence every minute for 15 minutes using the same excitation and emission. In this study, recombinant Drosophila atlastin is purified and reconstituted into preformed liposomes.The efficiency of the atlastin reconstitution is determined by floating reconstituted proteoliposomes in an iohexol discontinuous gradient. Samples of the top, middle, and bottom gradient layers are harvested and analyzed by SDS-PAGE and Coomassie staining. The quantification of the gel by densitometry shows a very high efficiency of reconstitution with negligible loses, with 96%of the total protein being found as proteoliposomes that floated to the top layer.Less than 1%of protein is unreconstituted and found in the middle layer, and only 3%is unreconstituted or aggregated and sedimented to the bottom layer. The orientation of atlastin after reconstitution is quantified by thrombin cleavage assays. Samples are analyzed with SDS-PAGE and Coomassie staining and quantified by densitometry.Most of the reconstituted protein is cleaved, with only 7%being protected from the protease. The kinetics and the extent of atlastin-mediated proteoliposome fusion are analyzed by lipid-mixing assays. An incubation of five minutes is performed before inducing fusion with GTP at the zero time point.After one hour, n-Dodecyl beta-D-maltoside was added to solubilize the proteoliposomes and get the maximum fluorescent resonance transfer release. The fusion maximum in the run is seen to be 11%of maximum fluorescence. Lipids are dissolved in chloroform.When preparing the lipid mixes, make sure to do this quickly, on a chemical hood, and on ice to avoid any evaporation. It is possible to analyze atlastin proteoliposomes by microscopy to visualize the effects of a membrane anchor on liposome shape and size. This method has been extended to study other membrane-anchored proteins and how the behave in a model lipid bilayer.This protocol uses titrated lipids to quantify lipid mixes. Make sure to take the necessary precautions when working with radioactive materials.

detergent-assisted-reconstitution-recombinant-drosophila-atlastin

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

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.

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 ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become the most widely used protein purification method in the modern literature. But, the application of affinity chromatography to metal binding proteins, especially those with redox sensitive metals such as iron, is often limited to laboratories with access to a glove box - equipment that is not routinely available in the undergraduate laboratory. In this article, we demonstrate our benchtop methods for isolation, IMAC purification and metal-ion reconstitution of a poly-histidine tagged, redox-active, non-heme iron binding extradiol dioxygenase and the assay of the dioxygenase with varied substrate concentrations and saturating oxygen. These methods are executed by undergraduate students and implemented in the undergraduate teaching and research laboratory with instrumentation that is accessible and affordable at primarily undergraduate institutions.

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

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 ...

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 ...

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a &#39;CAAX&#39; box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.

Prenylation is an important modification on peripheral membrane binding proteins. Insect cells can be manipulated to produce farnesylated and carboxymethylated KRAS4b in quantities that enable biophysical measurements of protein-protein and protein-lipid interactions

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...