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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Protein labeling

  1. Allow NHS-Rhodamine, 5-HT1A membrane fragments, and one 7 K MWCO spin desalting column to equilibrate at room temperature.
  2. Dissolve 1 mg of NHS-rhodamine in 100 µL of dimethyl sulfoxide (DMSO).
  3. Add 5 µL of 1 M sodium bicarbonate solution to increase the pH of 5-HT1AR solution to pH 8.
  4. Add 3.66 µL of the NHS-rhodamine solution to 50 µL of the 5-HT1AR solution and pipette gently up and down in a microcentrifug.......

Representative Results

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.......

Discussion

We have identified two steps that are critical to the success of the overall protocol:  plasma treatment and lipid deposition. Plasma cleaning of the coverslips is essential in ensuring that there is adequate coverage and adhesion of the agarose hydrogel to the glass coverslip. Plasma cleaning accomplishes two things: 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.......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)Avanti Polar Lipids, Alabaster, AL850375C-25mg
 TI-Eclipse inverted microscopeNikon, Melville, NYEclipse Ti
1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)Avanti Polar Lipids, Alabaster, AL850355C-25mg
13/16″ ID, 1″ OD silicon O-ringsSterling Seal & Supply, Neptune, IN5-003-8770
16-bit Cascade II 512 electron-multiplied charge coupled device cameraPhotometrics, Huntington Beach, CA Cascade II 512
1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)Avanti Polar Lipids, Alabaster, AL850457C-25mg
50 mW solid-state lasers at 488 nm and emission filter centered at 525 nm, and 561 nm with emission filter centered at 595 nmCoherent, Santa Clara, CA488/561-50-LS
5-HT1AR membrane fragmentsPerkin Elmer, Waltham, MARBHS1AM400UA
ATTO-488-1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)ATTO-TEC, Siegen, GermanyAD 488-155
Bench top plasma cleanerHarrick Plasma, Ithaca, NYPDC-32G
bovine serum albumin (BSA)Sigma Aldrich, St. Louis, MOA9418
chloroform (CHCl3)Millipore Sigma, Burlington, MACX1055
Cholesterol (Chol)Sigma Aldrich, St. Louis, MOC8667-5G
Corning 96-well Flat Clear BottomCorning, Corning, NY3904
Elmasonic E-Series E15H UltrasonicElma, Singen, Germany[no longer sold on main website]
glucoseSigma Aldrich, St. Louis, MOG7528
methanol (MeOH)Millipore Sigma, Burlington, MAMX0485
NanoDrop ND-1000Thermo Fisher Scientific, Waltham, MAND-1000
NHS-RhodamineThermo Fisher Scientific, Waltham, MA46406
phosphate buffered saline (PBS) (10x PBS)Corning, Corning, NY21-040
spinning-disc CSUX confocal headYokogawa,Tokyo, JapanCSU-X1
standard 25 mm no. 1 glass coverslipsChemGlass, Vineland, NJCLS-1760
sucroseSigma Aldrich, St. Louis, MOS7903
Sykes-Moore chambersBellco, Vineland, NJ1943-11111
Ultra-low melting temperature agaroseSigma Aldrich, St. Louis, MOA5030
VWR Analog HeatblockVWR International, Radnor, PA[no longer sold on main website]
VWR Tube RotatorVWR International, Radnor, PA10136-084
Zeba Spin Desalting Columns, 7K MWCO, 0.5 mLThermo Fisher Scientific, Waltham, MA89882

References

  1. Szoka, F., Papahadjopoulos, D. Comparative properties and methods of preparation of lipid vesicles (liposomes). Annual Review of Biophysics and Bioengineering. 9, 467-508 (1980).
  2. Mouritsen, O. G. Model answers to lipid membrane que....

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GUVsGiant Unilamellar VesiclesMembrane ProteinsGPCRsG Protein coupled Receptors5 HT1ARSerotonin ReceptorHydrogel ScaffoldLipid MembraneProtein IncorporationBiomimetic ModelIn Vitro Studies

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