We thank Matthew Blosser for valuable discussion and advice. This work was supported by the Office of Naval Research (N00014-16-1-2382) and the National Science Foundation (PHY-1915017).

1. Protein labeling
<ol>
	<li>Allow NHS-Rhodamine, 5-HT1A membrane fragments, and one 7 K MWCO spin desalting column to equilibrate at room temperature.</li>
	<li>Dissolve 1 mg of NHS-rhodamine in 100 &#181;L of dimethyl sulfoxide (DMSO).</li>
	<li>Add 5 &#181;L of 1 M sodium bicarbonate solution to increase the pH of 5-HT1AR solution to pH 8.</li>
	<li>Add 3.66 &#181;L of the NHS-rhodamine solution to 50 &#181;L of the 5-HT1AR solution and pipette gently up and down in a microcentrifug...

We have identified two steps that are critical to the success of the overall protocol:&#8239; plasma treatment and lipid deposition.&#8239;Plasma cleaning of the coverslips is essential in&#8239;ensuring that there is adequate coverage and adhesion of the agarose hydrogel to the glass coverslip. Plasma cleaning accomplishes two things:&#8239;first, it removes traces of organic matter from the glass surface; second, it activates the coverslip surface, allowing for an increase in wettability as the glass surface hydrophili...

Synthetic model membranes are powerful tools in the investigation of the fundamental properties and functions of biomembranes. Giant unilamellar vesicles (GUVs) are one of the most prominent platforms to study a variety of plasma membrane properties and can be engineered to mimic different physiological conditions1,2,3,4,5,6,7,8. It is well established that the plasma membrane and its organization play a key role in a multitude of cellular processes, such as signal transduction, adhesion, endocytosis, and transport9,10,11,12,13,14,15.
GUVs have been fabricated using various methods, including gentle hydration16, hydrogel swelling17, electroformation18, microfluidic techniques19,20,21,22, jetting23, and solvent exchange24,25,26. Due to challenges in handling integral membrane proteins (IMPs), in vitro platforms to study them have been limited. GUVs present a simplified platform for studying IMPs in an environment that mimics their native environment. Although there have been several approaches for protein reconstitution in GUVs, challenges arise from incorporating proteins with the correct orientation and maintaining protein functionality27.
Most successful protein-reconstitution in GUVs requires the detergent exchange method; which involves solubilizing the proteins from their native environment by detergents, followed by protein purification, and then replacing the detergent molecules with lipids through various methods28. While detergents serve to stabilize the tertiary structure of IMPs during purification, detergent micelles are a relatively unnatural environment for these proteins, which are better stabilized, particularly for functional studies, in lipid bilayers28,29,30. Moreover, incorporating functional transmembrane proteins into the lipid bilayer using traditional GUV fabrication techniques has been difficult due to the size, the delicacy of these proteins, and the additional detergent exchange steps that would be needed27,31,32,33. The use of organic solvent to remove detergents causes protein aggregation and denaturing34. An improved detergent-mediated method has been promising, however, caution is needed for the detergent removal step and optimization might be needed for specific proteins31,35. Additionally, methods that utilize electroformation could restrict the choice of protein and may not be suitable for all lipid compositions especially charged lipids31,36,37. Another technique that has been used is peptide-induced fusion of large unilamellar vesicles (LUVs) containing the desired protein with GUVs, though it was found to be laborious and can lead to the insertion of foreign molecules-the fusogenic peptides33,38,39. Giant plasma membrane vesicles (GPMVs), which are derived from living cells, can be used to overcome some of these issues, however they allow minimal control of the resultant lipid and protein composition14,40,41. Therefore, the integration of IMPs in the bilipid layer of GUVs using our modified agarose swelling method presents a reliable method to further examine these proteins in the membrane environment42,43,44,45.
Cellular signaling and communication involves a family of proteins known as G protein-coupled receptors (GPCRs); GPCRs are among the largest family of proteins and are associated with modulating mood, appetite, blood pressure, cardiovascular function, respiration, and sleep among many other physiological functions46. In this study, we used human serotonin 1A receptor (5-HT1AR) which is a prototypical member of the GPCR family. 5-HT1AR can be found in the central nervous system (CNS) and blood vessels; it influences numerous functions such as cardiovascular, gastrointestinal, endocrine functions, as well as participating in the regulation of mood47. A large barrier to GPCR research arises from their complex amphiphilic structure, and GUVs present a promising platform for the investigation of various properties of interest, ranging from protein functionality, lipid-protein interactions, and protein-protein interactions. Various approaches have been utilized to study lipid-protein interactions such as surface plasmon resonance (SPR)48,49, nuclear magnetic resonance spectroscopy (NMR)50,51, protein lipid overlay (PLO) assay51,52,53,54, native mass spectrometry55, isothermal titration calorimetry (ITC)56,57, and liposome sedimentation assay58,59. Our lab has used the simplified GUV approach to investigate the effect of lipid-protein interactions on protein functionality by incapsulating BODIPY-GTP&#947;S, which binds with the Gi&#945; subunit in the active state of the receptor. Their binding unquenches the fluorophore producing a fluorescence signal that could be detected over time45. Moreover, various studies investigated Lipid-protein interactions and the role of proteins in sensing or stabilizing membrane curvature60,61, and utilizing a feasible GUV approach could be a key advantage.
This protocol demonstrates a straightforward method to incorporate GPCRs into the membrane of GUVs using a modified agarose hydrogel system17,42. Furthermore, based on our previous work, our method could be suitable for IMPs that can bear short-term exposure to 30-40 &#176;C. Briefly, we spread a thin film of agarose combined with membrane fragments containing the GPCR of interest. Following gelation of this layer, we deposit a lipid solution atop the agarose and allow the solvent to evaporate. Rehydration of the system was then performed with an aqueous buffer, resulting in the formation of GUVs with protein incorporated in the lipid bilayer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td>Avanti Polar Lipids, Alabaster, AL</td>
			<td>850375C-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;TI-Eclipse inverted microscope</td>
			<td>Nikon, Melville, NY</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)</td>
			<td>Avanti Polar Lipids, Alabaster, AL</td>
			<td>850355C-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>13/16&#8243; ID, 1&#8243; OD silicon O-rings</td>
			<td>Sterling Seal &#38; Supply, Neptune, IN</td>
			<td>5-003-8770</td>
			<td></td>
		</tr>
		<tr>
			<td>16-bit Cascade II 512 electron-multiplied charge coupled device camera</td>
			<td>Photometrics, Huntington Beach, CA</td>
			<td>&#160;Cascade II 512</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)</td>
			<td>Avanti Polar Lipids, Alabaster, AL</td>
			<td>850457C-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mW solid-state lasers at 488 nm and emission filter centered at 525 nm, and 561 nm with emission filter centered at 595 nm</td>
			<td>Coherent, Santa Clara, CA</td>
			<td>488/561-50-LS</td>
			<td></td>
		</tr>
		<tr>
			<td>5-HT1AR membrane fragments</td>
			<td>Perkin Elmer, Waltham, MA</td>
			<td>RBHS1AM400UA</td>
			<td></td>
		</tr>
		<tr>
			<td>ATTO-488-1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)</td>
			<td>ATTO-TEC, Siegen, Germany</td>
			<td>AD 488-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench top plasma cleaner</td>
			<td>Harrick Plasma, Ithaca, NY</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin (BSA)</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform (CHCl3)</td>
			<td>Millipore Sigma, Burlington, MA</td>
			<td>CX1055</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol (Chol)</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>C8667-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96-well Flat Clear Bottom</td>
			<td>Corning, Corning, NY</td>
			<td>3904</td>
			<td></td>
		</tr>
		<tr>
			<td>Elmasonic E-Series E15H Ultrasonic</td>
			<td>Elma, Singen, Germany</td>
			<td>[no longer sold on main website]</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol (MeOH)</td>
			<td>Millipore Sigma, Burlington, MA</td>
			<td>MX0485</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop ND-1000</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>NHS-Rhodamine</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>46406</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline (PBS) (10x PBS)</td>
			<td>Corning, Corning, NY</td>
			<td>21-040</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning-disc CSUX confocal head</td>
			<td>Yokogawa,Tokyo, Japan</td>
			<td>CSU-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>standard 25 mm no. 1 glass coverslips</td>
			<td>ChemGlass, Vineland, NJ</td>
			<td>CLS-1760</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Sykes-Moore chambers</td>
			<td>Bellco, Vineland, NJ</td>
			<td>1943-11111</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low melting temperature agarose</td>
			<td>Sigma Aldrich, St. Louis, MO</td>
			<td>A5030</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Analog Heatblock</td>
			<td>VWR International, Radnor, PA</td>
			<td>[no longer sold on main website]</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Tube Rotator</td>
			<td>VWR International, Radnor, PA</td>
			<td>10136-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>89882</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction of model lipid membranes incorporating g-protein coupled receptors (gpcrs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

The concentration of protein was measured, and the degree of labeling was calculated as the molar ratio between the dye and the protein to be 1:1. By examining the GUVs using confocal microscopy, we were able to confirm successful formation and protein integration of the vesicles. The lipids were labeled with 0.4 mol% ATTO 488-DPPE, and the protein was covalently labeled via rhodamine NHS-ester modification of primary amines. Figure 2a and Figure 2b show a prote...

Watch this Scientific Journal Video about Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs at JoVE.com

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

Studying integral membrane proteins in their native environment has been a challenge due to the complexity of the plasma membrane.Giant unilamellar lipid vesicles, or GUVs, are one of the most prominent platforms to study biomembrane properties and they can be engineered to mimic various physiological conditions.This protocol describes a method to fabricate protein incorporated GUVs based on spontaneous incorporation, subsequent to hydrogel swelling.Traditional methods to fabricate GUVs are not generalizable for integral membrane protein incorporation.These methods are often low yield, methodologically complex and ad hoc, not applicable to broad classes of proteins.On the other hand, this protocol represents a flexible, robust and simple method to incorporate functional integral membrane proteins in GUVs.We accomplished this by preparing a hydrogel scaffold, containing the protein, and depositing a thin layer of lipid on top of it.In the example presented here, we incorporated G protein-coupled receptor, or GPCR, in the GUVs.Upon rehydration of the entire system, GUVs bud off of the surface of the hydrogel with the membrane containing properly oriented GPCRs.Allow all reagents to come to room temperature.During this time, clean the cover slips by placing them in methanol and sonicating for 30 minutes at 40

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Abstract