Abstract
SummaryThe β2-adrenoceptor (β2AR) is a well-established target in asthma and a prototypical G protein-coupled receptor for biophysical studies. Solubilization of membrane proteins has classically involved the use of detergents. However, the detergent environment differs from the native membrane environment and often destabilizes membrane proteins. Use of amphiphilic copolymers is a promising strategy to solubilize membrane proteins within their native lipid environment in the complete absence of detergents. Here we show the isolation of the β2AR in the polymer diisobutylene maleic acid (DIBMA). We demonstrate that β2AR remains functional in the DIBMA lipid particle and shows improved thermal stability compared with the n-dodecyl-β-D-maltopyranoside detergent-solubilized β2AR. This unique method of extracting β2AR offers significant advantages over previous methods routinely employed such as the introduction of thermostabilizing mutations and the use of detergents, particularly for functional biophysical studies.
Highlights
G protein-coupled receptors (GPCRs) are the largest family of membrane proteins within the human genome and are responsible for modulating a broad range of hormonal, neurological, and immune responses
We show the isolation of the b2AR in the polymer diisobutylene maleic acid (DIBMA)
We demonstrate that b2AR remains functional in the DIBMA lipid particle and shows improved thermal stability compared with the n-dodecyl-b-D-maltopyranoside detergent-solubilized b2AR
Summary
G protein-coupled receptors (GPCRs) are the largest family of membrane proteins within the human genome and are responsible for modulating a broad range of hormonal, neurological, and immune responses. The b2-adrenoceptor (b2AR) is a rhodopsin-like family GPCR (Schioth and Fredriksson 2005) and member of the adrenoceptor family, which signals primarily through coupling the heterotrimeric Gs protein. It is a well-established target for asthma and has become one of the most studied GPCRs with several structural (Wacker et al, 2010; Rasmussen et al, 2011; Bang and Choi 2015) and detailed biophysical studies (Manglik et al, 2015; Gregorio et al, 2017) into its activation mechanism. Multiple studies (Leitz et al, 2006; Whorton et al, 2007) have mimicked the native membrane environment and improved protein stability through reconstitution of membrane proteins in liposomes, amphipols, or synthetic nanodiscs; these all require initial use of detergents to extract the membrane protein from the membrane
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