The adenosine 2A receptor (A_2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A_2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A_2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A_2AR-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 A_2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp246^6.48 in TM6 and Trp268^7.33 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 231^6.33) to report on the dynamic cytoplasmic face of the A_2AR. 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 A_2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.

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