Ligand-Induced Conformational Changes in a SMALP-Encapsulated GPCR

Sarah J. Routledge, Mohammed Jamshad, Haydn A Little, Yu-Pin Lin, John Simms, Alpesh Thakker, Corrine M Spickett, Roslyn M Bill, Tim R. Dafforn, David R. Poyner, Mark Wheatley

    Research output: Contribution to journalArticlepeer-review

    21 Citations (Scopus)
    134 Downloads (Pure)


    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.
    Original languageEnglish
    Article number183235
    Number of pages37
    JournalBBA - Biomembranes
    Issue number6
    Early online date29 Feb 2020
    Publication statusPublished - 1 Jun 2020

    Bibliographical note

    Under a Creative Commons license


    • GPCR
    • SMALP
    • adenosine receptor
    • fluorescence


    Dive into the research topics of 'Ligand-Induced Conformational Changes in a SMALP-Encapsulated GPCR'. Together they form a unique fingerprint.

    Cite this