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 microcentrifuge tube. 
	NOTE: Ensure to have at least 10x molar excess of NHS-rhodamine.</li>
	<li>Keep the mixture protected from light and put on rotator at room temperature for 1 h.</li>
	<li>Wash a 7 k MWCO spin column with 200 &#181;L of 1x phosphate buffer saline (1x PBS) three times for 1.5 min at 1.5 RCF for each wash.</li>
	<li>Add the labeled protein to one column and balance the amount in another microcentrifuge tube.</li>
	<li>Spin down the labeled protein once for 5 min at 1.5 RCF.</li>
	<li>Take a UV-vis spectrum using a nanodrop spectrophotometer at 280 nm and 554 nm and calculate the labeling efficiency following the manufacturer&#39;s manual.</li>
	<li>Store the labeled protein covered at 5 &#176;C until further use. The solution is stable for approximately a week after labeling.</li>
</ol>
2. GUVs with membrane-incorporated 5-HT 1A
<ol>
	<li>Preparation of materials and reagents
	<ol>
		<li>Allow the protein, lipids and BSA (Bovine serum albumin) to equilibrate to room temperature.</li>
		<li>During this time, clean the coverslips by placing them in methanol and sonicating for 30 min at 40 &#176;C. Ensure that the methanol completely covers the coverslips and the water level in the water bath is above the level of the methanol in the container. 
		NOTE: Methanol is toxic and should be handled in appropriate chemical hood.</li>
		<li>Dry off the excess methanol on the coverslips with a gentle stream of air. Place the coverslip rack covered in a 40 &#176;C oven for 15 min to ensure that the excess coverslips dry off.</li>
		<li>Begin the plasma cleaning process. First, place the coverslips into the plasma cleaner and close off the air intake valve to evacuate all the air inside the chamber.</li>
		<li>Once the chamber is under vacuum, clean the coverslips for 5 min using high RF power setting and a near complete vacuum, with only a slight air intake into the vacuum chamber. To ensure the proper level of plasma, adjust the opening of the vacuum chamber such that the resultant color of the plasma is a steady, bright pink. 
		NOTE: It is crucial when using air that the plasma remains a bright pink color for the duration of the plasma treatment step, as a darker purple color indicates that there is an improper amount of air in the chamber and will result in a suboptimal plasma treatment.</li>
		<li>Once the 5 min have passed, shut off the RF power and release the vacuum. 
		NOTE: Upon removal from the plasma chamber, please ensure that the coverslips remain covered.</li>
	</ol>
	</li>
	<li>Hydrogel preparation
	<ol>
		<li>Combine 6 mg of ultra-low melting temperature agarose with 300 &#181;L of ultrapure water (i.e., 2% (w/v) agarose). 
		NOTE: 2% agarose will be used to make protein-free GUVs. Agarose solution can be kept at 45 &#176;C for two days.</li>
		<li>Combine 9 mg of ultra-low temperature agarose with 300 &#181;L of ultrapure water for 3 w/v% agarose by as prepared in step 3.1. 3% agarose will be used to make protein incorporated GUVs.</li>
		<li>Vortex the solution briefly before placing them on the 90 &#176;C heat block for 10 min. Then, vortex the tube again before transferring it to a 45 &#176;C heat block to keep it in the molten form until further use.</li>
	</ol>
	</li>
	<li>Agarose and protein mixing
	<ol>
		<li>Mix 21 &#181;L of 3% agarose with the 7 &#181;L of 5-HT1AR membrane fragments. Pipette up and down slowly many times to ensure adequate mixing. Then, incubate at 45 &#176;C for 1 min.</li>
	</ol>
	</li>
	<li>Hydrogel and lipid deposition
	<ol>
		<li>For protein-free GUVs: Make a thin film on freshly plasma-cleaned coverslips using 20 &#181;L of 2% agarose. Quickly drop another coverslip on top of the agarose droplet and gently slide the coverslips apart to make a thin film on both coverslips. 
		NOTE: This step is tricky in that the sliding of the droplet must occur while the agarose is still in the molten form.</li>
		<li>For protein-incorporated GUVs: Pipette the protein/agarose mixture up and down one more time, and then deposit 20 &#181;L of the 2% agarose on a plasma-cleaned coverslip. Follow the slip-casting directions as described above.</li>
		<li>Allow the agarose to gel protected from light for 30 min at room temperature.</li>
		<li>Deposit the lipids dropwise on top of the agarose layer. Use a total of 10 &#181;L of 2 mg/mL of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with 0.4 Mol% 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) labeled with ATTO 488 (ATTO-488-DPPE) (or lipid mixture of interest) in chloroform on top of the agarose film. Deposit the droplets using a gas chromatography needle and spread one droplet at a time around via a gentle air stream. 
		NOTE: Caution is needed with this step to make a relatively uniform layer of lipids on top of the hydrogel. Also, chloroform is toxic and should be handled in appropriate chemical hood.</li>
		<li>Assemble the Sykes-Moore (S-M) chambers by placing an O-ring on top of the coverslip, and then placing the top component of the chamber on top of the O-ring. Use the key provided by the manufacturer to assemble the chamber by screwing the chamber components together to seal the chamber and prevent any leakage. 
		NOTE: The top of the chamber should be tightened on the O-ring but caution is needed to ensure the coverslip stays intact as the coverslip can crack if the O-ring doesn&#39;t sit properly in the chamber. Also, ensure that the chamber is sealed tight enough such that the chamber does not leak when the swelling solution is added. Failure to tighten the chamber enough will result in leaks and loss of sample.</li>
	</ol>
	</li>
	<li>Swelling and harvesting of vesicles
	<ol>
		<li>Hydrate the entire system by gently pipetting 450 &#181;L of 200 mM sucrose in 1x PBS and gently tapping the chambers to ensure adequate buffer coverage of the hydrogel-lipid layers. 
		NOTE: The sucrose solution can be replaced with a rehydration buffer containing biological probes of interest.</li>
		<li>Place the chambers at 45 &#176;C and cover the top part of the chamber with a coverslip to prevent evaporation. Allow the sample to swell, protected from debris and light for 1 h.</li>
		<li>Add 100 &#181;L of 1 mg/mL BSA in ultrapure water into each well of a 96-well plate intended to be used. Incubate at room temperature for 1 h.</li>
		<li>Wash three times with ultra-pure water and once with 200 mM sucrose in 1x PBS.</li>
		<li>Finally, add 200 mM of glucose in 1x PBS until the addition of the GUV sample solution. 
		NOTE: BSA was used to block GUV adsorption.</li>
		<li>After allowing the hydrogel to swell, gently shake and tap the chamber to dislodge any GUVs that may remain attached to the hydrogel surface. Then, carefully pipette up the GUV-sucrose solution. 
		NOTE: As an optional step to ensure all vesicles are detached from the surface, gently pipette some of the sucrose suspension back onto the hydrogel surface.</li>
		<li>Move the suspension into a previously prepared microcentrifuge tube containing 700 &#181;L of 200 mM glucose in 1x PBS. 
		NOTE: The density gradient will lead to settling of the vesicles to the bottom of the centrifuge tube.</li>
		<li>Allow the vesicles to settle for another hour to ensure that the vesicles can sink to the bottom of the microcentrifuge tube, allowing for optimal collection.</li>
		<li>After the settling of GUVs in glucose, transfer 300 &#181;L from the bottom of the centrifuge tube (the settled vesicles) into the previously prepared and BSA-treated 96-well plate to examine the vesicles under the confocal microscope. 
		NOTE: Be sure to avoid the very bottom of the microcentrifuge tube to minimize the amount of debris collected in the final sample.</li>
	</ol>
	</li>
	<li>Check the samples under the microscope.
	<ol>
		<li>Shine a 488 nm laser on the sample (that allows us to visualize the membrane, as the bilayer has been labeled with ATTO-488-DPPE).</li>
		<li>Shine a 561 nm laser on the sample (that allows us to visualize the protein, since it has been labeled with NHS-Rhodamine). 
		&#8203;NOTE: Caution is needed while imaging the sample as photooxidation can destabilize the vesicles. Vesicles were observed on the same day.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62830/62830fig01.jpg" /> 
Figure 1: Illustration of the detailed protocol steps. Created with BioRender.com <a href="https://www.jove.com/files/ftp_upload/62830/62830fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The authors have no conflicts of interest to disclose.

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 ...

construction-model-lipid-membranes-incorporating-g-protein-coupled

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3.4K Views.  University of Southern California. 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.

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

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 ...

Biochemistry

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

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 ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

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

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

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

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

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

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

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

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

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Biology

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

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

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

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

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