title: G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent.
abstract: G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.
authors.affiliation: *School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Jamshad
authors.affiliation: *School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.
authors.forename: Jack
authors.initials: J
authors.lastname: Charlton
authors.affiliation: *School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.
authors.forename: Yu-Pin
authors.initials: YP
authors.lastname: Lin
authors.affiliation: †School of Life and Health Sciences, Aston University, Birmingham B4 7ET, U.K.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Routledge
authors.affiliation: †School of Life and Health Sciences, Aston University, Birmingham B4 7ET, U.K.
authors.forename: Zharain
authors.initials: Z
authors.lastname: Bawa
authors.affiliation: ‡School of Cancer Studies, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Knowles
authors.affiliation: ‡School of Cancer Studies, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.
authors.forename: Michael
authors.initials: M
authors.lastname: Overduin
authors.affiliation: §Discovery Sciences, AstraZeneca R&D Mölndal, 43183 Mölndal, Sweden.
authors.forename: Niek
authors.initials: N
authors.lastname: Dekker
authors.affiliation: *School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.
authors.forename: Tim R
authors.initials: TR
authors.lastname: Dafforn
authors.affiliation: †School of Life and Health Sciences, Aston University, Birmingham B4 7ET, U.K.
authors.forename: Roslyn M
authors.initials: RM
authors.lastname: Bill
authors.affiliation: †School of Life and Health Sciences, Aston University, Birmingham B4 7ET, U.K.
authors.forename: David R
authors.initials: DR
authors.lastname: Poyner
authors.affiliation: *School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.
authors.forename: Mark
authors.initials: M
authors.lastname: Wheatley
mesh_terms: D002460:Cell Line; D057809:HEK293 Cells; D006801:Humans; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry; D010843:Pichia / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D043705:Receptor, Adenosine A2A / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
chemical_list: D043705:Receptor, Adenosine A2A; D011994:Recombinant Proteins; C411362:dodecyl maltopyranoside; D008320:Maltose