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Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)
Chapters
Summary February 5th, 2022
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.
Transcript
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
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