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. Preparación de liposomas Andamios NOTA: Idealmente, los experimentos iniciales se debe utilizar un andamio relativamente simple y sin rasgos (compuesto por DOPC (1,2-sn dioleoyl- glicero-3-fosfocolina o PC) y DGS-NTA (Ni2 +) (1,2-dioleoil - sn -glicero-3 - [(N - (5-amino-1-carboxipentil) iminodiacético) succinil]. (sal de níquel)) Building fuera de estos experimentos, la carga de los lípidos, la flexibilidad, y la curvatura puede introducirse como f...

<ol>
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Este protocolo ofrece un método para la investigación de las interacciones proteína-proteína que participa en proteínas integrales de membrana. Mediante la utilización de un andamio modular de liposomas, los investigadores son capaces de evaluar la actividad de una o más proteínas en un entorno de lípidos proximal. Estudios anteriores han demostrado un método similar para las enzimas de los receptores de la membrana plasmática 24-26. Hemos ampliado este método para incorporar...

El estudio de las interacciones proteína-proteína de membrana proximal es una tarea difícil debido a la dificultad para recapitular el entorno natural de las proteínas integrales de membrana que participan 1. Esto es debido a la necesidad de solubilización de detergente y la orientación inconsistente de proteínas en proteoliposomas. Con el fin de evitar estos problemas, se ha empleado una estrategia en la que dominios solubles de proteínas integrales de membrana se expresan como proteínas de fusión His-tag, y estos fragmentos solubles están anclados a los liposomas de andamio a través de interacciones con NTA (Ni 2+) grupos de cabeza en el lípido superficie. El uso de estos andamios, las interacciones proteína-lípido proximal pueden ser investigados en un rango de composiciones de lípidos y proteínas. Hemos aplicado con eficacia este método para investigar las interacciones proteína-proteína críticos que rigen el ensamblaje del complejo de la fisión mitocondrial y examinar las interacciones de lípidos que modulan este proceso 2. Durante la fisión mitocondrial, una proteína de la remodelación de membrana conservada, denominado proteína relacionada con dynamin 1 (Drp1) 3, es reclutado para la superficie de la externa mitocondrial membrana (OMM) en respuesta a señales celulares que regulan la homeostasis de la energía, la señalización de apoptosis, y varias otras procesos mitocondriales integrales. Este GTPasa grande, citosólica es reclutado a la superficie de la mitocondria a través de interacciones con proteínas integrales de OMM 4 - 8. El papel de una de tales proteínas, Fisión mitocondrial Factor (MFP), ha sido difícil de dilucidar debido a una débil interacción aparente con Drp1 in vitro. Sin embargo, los estudios genéticos han demostrado claramente que Mff es esencial para el éxito de 7,8 fisión mitocondrial. El método descrito en este manuscrito fue capaz de superar las deficiencias anteriores mediante la introducción de lípidos interacciones simultáneas que promueven interacciones Drp1-MFP. En general, este novedoso ensayo REVEAconducido interacciones fundamentales ensamblaje del complejo de fisión mitocondrial de guía y proporcionó una nueva etapa para estudios estructurales y funcionales en curso de esta máquina molecular esencial. Hasta la fecha, el examen de las interacciones entre Drp1 y Mff se han complicado por la flexibilidad inherente de Mff 9, la heterogeneidad de Drp1 Polímeros 2,10, y la dificultad en la purificación y reconstitución de longitud completa Mff con un dominio transmembrana intactas 11. Hemos abordado estos problemas mediante el uso de liposomas NTA (Ni2 +) de andamios para reconstituir His-Mff que carece de su dominio transmembrana (MffΔTM-Su 6). Esta estrategia fue una ventaja, ya MffΔTM era extremadamente soluble cuando se sobre-expresan en E. coli, y esta proteína aislada se reconstituyó fácilmente en liposomas de andamios. Cuando atado a estas plantillas de lípidos, Mff asumió una, la orientación hacia el exterior idéntica en la superficie de la membrana.Además de estas ventajas, los lípidos mitocondriales, tales como cardiolipina, se añadieron a estabilizar Mff plegado y la asociación con la membrana 11. La cardiolipina también interactúa con el dominio variable de Drp1 2,12 que puede estabilizar esta región desordenada y facilitar el montaje de la maquinaria de fisión. Este método robusto es ampliamente aplicable para futuros estudios que pretenden evaluar las interacciones proteína-membrana proximal. Mediante el uso de interacciones adicionales tethering / afinidad, la sofisticación de estos estudios de reconstitución de membrana se puede mejorar para imitar la complejidad adicional que se encuentra en la superficie de las membranas dentro de las células. Al mismo tiempo, las composiciones lipídicas se pueden modificar para imitar con mayor precisión los ambientes nativos de estos complejos macromoleculares. En resumen, este método proporciona un medio para examinar las contribuciones relativas de las proteínas y lípidos de membrana en la configuración de morfologías que durante proc celulares críticaseses.

<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>
			<td style="width:136px;">456-1096</td>
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using scaffold liposomes to reconstitute lipid-proximal protein-protein interactions in vitro

Los estudios de proteínas integrales de membrana in vitro con frecuencia se complica por la presencia de un dominio transmembrana hidrófobo. Para complicar aún más estos estudios, la reincorporación de proteínas de membrana solubilizada con detergente en los liposomas es un proceso estocástico donde topología de la proteína es imposible de aplicar. Este trabajo ofrece un método alternativo para estas técnicas desafiantes que utiliza un andamio a base de liposomas. solubilidad de la proteína se ve reforzada por la supresión del dominio transmembrana, y estos aminoácidos se sustituyen con un resto de anclaje, tal como una etiqueta de His. Esta correa de sujeción interactúa con un grupo de anclaje (Ni 2+ coordinado por ácido nitrilotriacético (NTA (Ni 2+)) para marcado con His proteínas), que hace cumplir una topología de proteína uniforme en la superficie del liposoma. Se presenta un ejemplo en el que la interacción entre la proteína relacionada con dynamin 1 (Drp1) con una proteína de membrana, la fisión mitocondrial Factor (MFP), fue investigated utilizando este método andamio liposoma. En este trabajo, hemos demostrado la capacidad de reclutar Mff eficiente Drp1 solubles a la superficie de los liposomas, que estimuló su actividad GTPasa. Por otra parte, Drp1 pudo tubulate la plantilla de lípidos decorados a Mff en presencia de lípidos específicos. Este ejemplo demuestra la eficacia de los liposomas de andamio usando ensayos estructurales y funcionales y destaca el papel de Mff en la regulación de la actividad Drp1.

Mientras que la interacción entre Drp1 y Mff ha demostrado ser importante para la fisión mitocondrial, esta interacción ha sido difícil de recapitular in vitro. Nuestro objetivo era emular mejor el entorno celular en el que Drp1 y Mff interactúan. Con este fin, los liposomas que contienen concentraciones limitantes de NTA (Ni 2+) grupos de cabeza se prepararon mediante rehidratación de una película de lípidos como se describió anteriormente. La solución de l...

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.

El uso de andamios liposomas para reconstituir las interacciones proteína-proteína-lípidos proximal<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

La anisotropía de fluorescencia como una herramienta para estudiar las interacciones proteína-proteína

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

Investigación

Bioquímica

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

Resuelta en el tiempo de ionización por electrospray de hidrógeno-deuterio Espectrometría de Masas de cambio para el estudio de la estructura proteica y Dinámica

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

Péptidos de penetración celular biotinilado para estudiar interacciones de proteínas intracelulares

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

Uso del traste de una sola molécula a estudiar la correlación de las interacciones proteína

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

Un análisis de espectroscopia de fluorescencia fluctuación de interacciones de proteínas en los contactos célula-célula

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

Mutagénesis sitio-dirigida para In Vitro e In Vivo experimentos, ejemplificados con las interacciones de la ARN en 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

Aplicación de la interferometría de Biolayer (BLI) para el estudio de las interacciones proteínas y proteínas en la transcripción

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

Evaluación de interacciones proteínas-proteínas utilizando una técnica de digestión en la membrana

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

Espectroscopia de correlación cruzada de fluorescencia de doble color para estudiar la interacción proteína-proteína y la dinámica de proteínas en células vivas

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

Ensayo pulldown junto con coexpresión en células bacterianas como una herramienta eficiente en el tiempo para probar interacciones desafiantes proteína-proteína

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