Name: using scaffold liposomes to reconstitute lipid-proximal protein-protein interactions in vitro
Uploaded: 2017-01-11T12:30:00
Description: Watch this Scientific Journal Video about Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro at JoVE.com

The authors would like to acknowledge the funding received from the American Heart Association (SDG12SDG9130039).

1. Steiger liposoompreparaat OPMERKING: Idealiter eerste experimenten een relatief eenvoudige en eentonige steiger (bestaande uit DOPC (1,2-dioleoyl- sn-glycero-3-fosfocholine of PC) en DGS-NTA (Ni 2+) (1,2-dioleoyl gebruiken - sn-glycero-3 - [(N - (5-amino-1-carboxypentyl) iminodiazijnzuur) succinyl]. (nikkelzout)) Het is gebaseerd op deze proeven lipide lading, flexibiliteit en kromming kan worden ingebracht als individuele factoren met de mogelijkheid o...

<ol>
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Dit protocol biedt een methode voor het onderzoeken van eiwit-eiwit interacties van integrale membraaneiwitten. Met behulp van een liposoom modulaire steiger, onderzoekers zijn geschikt om de activiteit van één of meer eiwitten in een lipide-proximale omgeving. 26 - Eerdere studies hebben een soortgelijke werkwijze voor receptor enzymen van de plasmamembraan 24 aangetoond. We hebben deze methode om lipide co-factoren op te nemen en interacties tussen eiwitten die deel uitmaken van de m...

Bestuderen membraanproximale eiwit-eiwit interacties is een uitdagende poging door problemen als gevolg indient de natuurlijke omgeving van het integrale membraaneiwitten betrokken 1. Dit komt door de noodzaak detergens oplosbaar en inconsistente oriëntatie van eiwitten in proteoliposomen. Om deze problemen te voorkomen, hebben we een strategie waarbij oplosbare domeinen van integrale membraaneiwitten zijn uitgedrukt His-tag fusie-eiwitten toegepast, en deze oplosbare fragmenten zijn verankerd aan scaffold liposomen via interacties met NTA (Ni2 +) kopgroepen van het lipide oppervlak. Met deze steigers, kunnen lipide-proximale eiwitinteracties worden onderzocht over een traject van lipiden en eiwitten samenstellingen. Wij hebben effectief pasten deze methode het kritische eiwit-eiwit interacties die assemblage van de mitochondriale complex splitsing regelt onderzoeken en onderzoeken lipide interacties die deze pr modulerenocess 2. Tijdens mitochondriale splitsing, een geconserveerd membraan remodeling eiwit, genaamd-Dynamin gerelateerd eiwit 1 (Drp1) 3 wordt aangetrokken naar het oppervlak van de buitenste mitochondriale membraan (OMM) in reactie op cellulaire signalen die energie homeostase, apoptotische signalering, en verscheidene andere reguleren integraal mitochondriale processen. Deze grote cytosolische GTPase wordt aangetrokken naar het oppervlak van mitochondria door interactie met geïntegreerde OMM eiwitten 4-8. De rol van een dergelijk eiwit, Mitochondriale Splitsing Factor (MFF), moeilijk op te helderen als gevolg van een schijnbare zwakke interactie met Drp1 in vitro. Desondanks hebben genetische studies duidelijk aangetoond dat MFK essentieel voor een succesvolle mitochondriale kernsplijting 7,8. De methode in dit manuscript beschreven was in staat om eerdere tekortkomingen te verhelpen door de invoering van gelijktijdige lipide interacties die Drp1-MFF interacties te promoten. Over het algemeen, deze roman assay REVEAgeleid fundamentele interacties begeleiden van assemblage van het mitochondriale kernsplijting complex en voorzien van een nieuwe fase voor de lopende structurele en functionele studies van deze essentiële moleculaire machine. Tot op heden zijn onderzocht interacties tussen Drp1 en MFF bemoeilijkt door de inherente flexibiliteit van MFF 9, de heterogeniteit van Drp1 polymeren 2,10, en de moeilijkheid bij het zuiveren en reconstitutie full-length MFF met intact transmembraandomein 11. We gericht deze uitdagingen met behulp van NTA (Ni 2+) steiger liposomen His-tag MFF reconstrueren ontbreekt haar transmembraandomein (MffΔTM-His 6). Deze strategie is voordelig omdat MffΔTM was zeer oplosbaar toen over-uitgedrukt in E. coli, en deze geïsoleerde eiwit was gemakkelijk gereconstitueerd op steiger liposomen. Wanneer deze gebonden lipiden sjablonen, MFF verondersteld een identieke, naar buiten gerichte oriëntatie op het oppervlak van het membraan.Naast deze voordelen, mitochondrial lipiden, zoals cardiolipine, werden toegevoegd aan MFF vouwen en associatie stabiliseren het membraan 11. Cardiolipine ook interactie met het variabele domein van Drp1 2,12 waarvan de ongeordende gebied kan stabiliseren en assemblage van de splitsing machine te vergemakkelijken. Deze robuuste methode is breed toepasbaar voor toekomstige studies die streven naar het membraan-proximale eiwit interacties te evalueren. Door het gebruik van aanvullende tethering / affiniteit interacties, kan de complexiteit van deze membraan reconstitutie studies worden verbeterd om verder compliceren vinden op het oppervlak van membranen in cellen nabootsen. Tegelijkertijd kunnen lipidesamenstellingen worden gemodificeerd om nauwkeuriger na te bootsen de natuurlijke omgeving van deze macromoleculaire complexen. Kortom, deze werkwijze verschaft een middel om de relatieve bijdragen van eiwitten en lipiden in vormgeving membraan morfologie om tijdens kritieke cellulaire proc onderzochtsen.

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Phosphatidylcholine (DOPC)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">850375</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Phosphatidylethanolamine (DOPE)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">850725</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:287px;">DGS-NTA(Ni2+)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">790404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Bovine Heart Cardiolipin (CL)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">840012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Chloroform</td>
			<td style="width:229px;">Acros Organics</td>
			<td style="width:136px;">268320010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Liposome Extruder</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">610023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Cu/Rh Negative Stain Grids</td>
			<td style="width:229px;">Ted Pella</td>
			<td style="width:136px;">79712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microfuge Tube</td>
			<td style="width:229px;">Beckman</td>
			<td style="width:136px;">357448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">GTP</td>
			<td style="width:229px;">Jena Biosciences</td>
			<td style="width:136px;">NU-1012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">GMP-PCP</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">M3509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microtiter Plate strips</td>
			<td style="width:229px;">Thermo Scientific</td>
			<td style="width:136px;">469949</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">EDTA</td>
			<td style="width:229px;">Acros Organics</td>
			<td style="width:136px;">40993-0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Instant Blue Coomassie Dye</td>
			<td style="width:229px;">Expedeon</td>
			<td style="width:136px;">ISB1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">HEPES</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:136px;">BP310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">BME</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">M6250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">KCL</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:136px;">P330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">KOH</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:136px;">P250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Magnesium Chloride</td>
			<td style="width:229px;">Acros Organics</td>
			<td style="width:136px;">223211000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">4-20% SDS-PAGE Gel</td>
			<td style="width:229px;">Bio Rad</td>
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using scaffold liposomes to reconstitute lipid-proximal protein-protein interactions in vitro

Studies van integrale membraaneiwitten in vitro worden vaak gecompliceerd door de aanwezigheid van een hydrofoob transmembraandomein. Verdere complicerende deze studies, heropname van wasmiddel-opgeloste membraaneiwitten in liposomen is een stochastisch proces waarbij eiwitten topologie is onmogelijk om af te dwingen. Dit document biedt een alternatieve methode om deze moeilijke techniek die een liposoom gebaseerde scaffold gebruikt. Eiwit oplosbaarheid verbeterd door deletie van het transmembraandomein, en deze aminozuren zijn vervangen door een tethering groep, zoals een His-tag. Deze tuier interageert met een verankerende groep (Ni2 + gecoördineerd door nitrilotriazijnzuur (NTA (Ni2 +)) voor His-gelabelde eiwitten), waarbij een uniforme eiwit topologie dwingt aan het oppervlak van het liposoom. Een voorbeeld wordt gepresenteerd, waarin de interactie tussen-Dynamin gerelateerd eiwit 1 (Drp1) met een integraal membraaneiwit, Mitochondriale Fission Factor (MFK), was investigated met behulp van deze steiger liposoom methode. In dit werk hebben we het vermogen van MFK oplosbare Drp1 efficiënt rekruteren op het oppervlak van liposomen, waarvan de GTPase activiteit gestimuleerd aangetoond. Bovendien Drp1 was in staat om het MFK-versierde lipide-template in de aanwezigheid van specifieke lipiden buisvormig maken. Dit voorbeeld toont de effectiviteit van scaffold liposomen middels structurele en functionele assays en wordt de rol in het reguleren van MFF Drp1 activiteit.

Terwijl de interactie tussen Drp1 en MFF is aangetoond belangrijk mitochondriale splitsing zijn, heeft deze interactie moeilijk te herhalen in vitro. Ons doel was om beter na te bootsen de cellulaire omgeving waarin Drp1 en MFF interactie. Daartoe liposomen die limietconcentraties van NTA (Ni2 +) kopgroepen werden bereid door hydrateren van een vetfilm zoals hierboven beschreven. De lipideoplossing bestaat aanvankelijk van unilamellaire vesicles en multilamellaire van...

Watch this Scientific Journal Video about Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro at JoVE.com

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.

Gebruik Steiger Liposomen lipide-proximale eiwit-eiwit interacties reconstitueren<em&gt; In Vitro</em

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

The overall goal of this technique is to capture lipid-proximal protein-protein interactions in vitro. This method can help answer questions in the mitochondrial dynamics field, such as how protein-protein interactions at the mitochondrial outer membrane can be affected by accessory lipids. The main advantage of this technique is that tethering soluble protein domains to lipid templates ensures that a more native confirmation can be achieved.Moreover, complex interactions between soluble proteins and their tethered partners can be assessed in the presence of defined lipid cofactors. Though we use this method to investigate protein interactions in the mitochondrial fission machinery, it can also be applied to other protein complexes that localize to diverse membrane compartments. Demonstrating this procedure will be Ryan, a grad student from my laboratory.First, combine chloroform solutions of the desired lipids in a clean glass test tube. Evaporate the solvent with dry nitrogen gas while rotating the tube to form a thin lipid film. Then remove the residual solvent with the centrifugal evaporator for one hour at 37 degrees Celsius.Following this, add pre-heated buffer A to give a final lipid concentration of one to two millimolar. Incubate for 30 minutes at 37 degrees Celsius with occasional vortexing to fully resuspend the lipid mixture. After incubation, transfer the lipid mixture to a plastic test tube and place the tube in liquid nitrogen for approximately 30 seconds until completely frozen.Then place the tube in a 37 degree Celsius waterbath for one to two minutes until fully thawed. Next prepare a lipid extruder by soaking four filter supports and a polycarbonate filter in buffer A and assembling the extruder according to the manufacturer's instructions. Extrude the lipid solution through the filter 21 times using gentle constant pressure to ensure a homogenous size distribution.While extruding, use slow constant pressure to ensure liposome size homogeneity. With a one micron filter, this is less important. But with smaller filter sizes, can introduce more apparent heterogeneity.Store the extruded liposomes at four degrees Celsius. Incubate His-tagged mitochondrial fission factor or Mff with scaffold liposomes for at least 15 minutes at room temperature in buffer A and BME. For an Mff-free control, incubate the liposomes with a His-tagged control protein, such as GFP, to bind and shield exposed Nickel-NTA.Add dynamin-related protein one or Drp1 to the liposome mixture and incubate for one hour at room temperature. Next, transfer five microliters of the sample to a sheet of laboratory film and lay a carbon-coated copper-rhodium grid on top of the sample. After incubating for one minute, remove the excess liquid with filter paper and add a drop of 2%uranyl acetate.After incubating for another minute, remove the excess stain with filter paper, and transfer the sample to a grid box. On the following day, image the samples using a transmission electron microscope at the 18, 500 to 30, 000X magnification to observe ultrastructural changes in protein and liposome morphologies. Incubate His-tagged Mff and Fis1 with the scaffold liposomes for 15 minutes at room temperature in buffer A and BME.Add Drp1 and incubate each mixture for an additional 15 minutes at room temperature. Transfer the tubes to the thermocycler set to 37 degrees Celsius and initiate the reactions by adding GTP and magnesium chloride. At the desired time points, transfer 20 microliters of each reaction mixture to wells of a microtiter plate containing five microliters of 0.5 molar EDTA to chelate the magnesium and stop the reaction.Following this, prepare a set of phosphate standards by diluting potassium phosphate in buffer A and BME to calibrate the results. Add 20 microliters of each standard to the wells containing five microliters of 0.5 molar EDTA. Add 150 microliters of malachite green reagent to each well.Finally, read the OD 650 five minutes after each addition. GTP will hydrolyze slowly in the presence of the acidic malachite green reagent. So this reagent should be added to all sample wells within 10 minutes of measuring the absorbance to maximize signal to noise.In the absence in Drp1, neither Mff nor GFP resulted in membrane deformation. And only featureless liposomes were observed for GFP-decorated enriched scaffold liposomes or ESL. However, when Drp1 was added to Mff-decorated ESL templates remodeling of the liposomes was evident.Decoration of the SL with Mff enhanced GTPase activity. Conversely, when the exposed NTA head groups were blocked with His-tagged GFP, this augmented GTPase activity was ablated. Tethering of Fis1 lacking its transmembrane domain to the SL, also failed to elicit a stimulation of Drp1's GTPase activity.Similar to the SL, addition of SL/Cl to Drp1 resulted in a slight stimulation of GTPase activity that was reversed by tethering His-tagged Fis1 or GFP to the liposomes. A synergy between Mff and cardiolipin was observed as the GTPase activity of Drp1 was stimulated 2.6-fold when it was incubated with Mff-decorated SL/CL. When SL/PE/CL templates were decorated with Mff, Drp1 activity was enhanced.The ability of Drp1 to remodel liposomes into lipid tubules was enhanced by the addition of PE to the scaffold liposomes. Once mastered, these scaffolds can be prepared and used in hours if the procedure is performed properly. Following this procedure, other protein and lipid interaction methods like sedimentation and light-scattering assays can be performed in order to characterize functional interactions.The application of this technique allows people examining protein translocation to a defined biological membrane to explore transient protein interactions and the impact of specific lipid cofactors in the formation and stabilization of essential cellular assemblies.

Research

JoVE Journal

Biochemistry

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

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Fluorescentie anisotropie als een instrument om eiwit-eiwit interacties te bestuderen

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Tijdsgeresolveerde elektrosprayionisatie waterstof-deuterium Exchange Massaspectrometrie voor het bestuderen van Protein Structure en Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Biotinyleerd cel-penetrating Peptides te bestuderen van intracellulaire eiwit-eiwitinteractie

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Met behulp van 3 Single-molecuul FRET te bestuderen de correlatie van eiwitinteractie

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Een fluorescentie schommelingen spectroscopie Assay voor eiwit-eiwitinteractie op cel contacten

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Plaats-geleide mutagenese voor In Vitro en In Vivo experimenten wordt geïllustreerd met RNA interacties in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Toepassing van Biolayer interferometrie (BLI) voor het bestuderen van eiwit-eiwit interacties in transcriptie

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Evaluatie van eiwit-eiwit interacties met behulp van een on-membraan Vergistings techniek

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

Dual-Color Fluorescence Cross-Correlation Spectroscopy om eiwit-eiwitinteractie en eiwitdynamica in levende cellen te bestuderen

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown-test in combinatie met co-expressie in bacteriecellen als een tijdefficiënt hulpmiddel voor het testen van uitdagende eiwit-eiwitinteracties

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...