Ligand-induced conformational changes in a SMALP-encapsulated GPCR.


The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A2AR-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp2466.48 in TM6 and Trp2687.33 at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 2316.33) to report on the dynamic cytoplasmic face of the A2AR. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.

Publication DOI:
Divisions: College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences > Aston Pharmacy School
Additional Information: © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( Funding: This work was supported by funding from the Biotechnology and Biological Sciences Research Council; grant numbers BB/I020349/1 (to MW and TRD), BB/I019960/1 (to DRP and RMB), BB/R016615/1 (to MW and TRD) and BB/R016755/1 (to DRP). HAL was supported by a BBSRC-MIBTP award.
Uncontrolled Keywords: Adenosine receptor,Fluorescence,GPCR,SMALP,Biophysics,Biochemistry,Cell Biology
Publication ISSN: 1879-2642
Full Text Link:
Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
PURE Output Type: Article
Published Date: 2020-06-01
Published Online Date: 2020-02-29
Accepted Date: 2020-02-26
Authors: Routledge, Sarah J.
Jamshad, Mohammed
Little, Haydn A.
Lin, Yu Pin
Simms, John (ORCID Profile 0000-0002-4675-0902)
Thakker, Alpesh
Spickett, Corinne M. (ORCID Profile 0000-0003-4054-9279)
Bill, Roslyn M. (ORCID Profile 0000-0003-1331-0852)
Dafforn, Tim R.
Poyner, David R. (ORCID Profile 0000-0003-1590-112X)
Wheatley, Mark



Version: Published Version

License: Creative Commons Attribution

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