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.

中文翻译：

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SMALP封装的GPCR中配体诱导的构象变化。

将G蛋白偶联受体（GPCR）腺苷2A受体（A2AR）溶解并纯化，封装在苯乙烯马来酸脂质颗粒（SMALPs）中。纯化的A2AR-SMALP与巴斯德毕赤酵母（Pichia pastoris）的质膜特征性磷脂有关，该毕赤酵母用于表达该宿主，证实了A2AR-SMALP包裹了天然脂质。A2AR-SMALP的荧光光谱显示出由内源性Trp残基产生的在330 nm处的特征性宽发射峰。反向激动剂ZM241385导致荧光发射增加30％，通常伴随着发射波长的红移。发射光谱还显示出在321 nm，335 nm和350 nm处的亚峰，表明添加ZM241385后单个Trp居住在不同的环境中。激动剂NECA对A2AR-SMALP荧光光谱没有影响。Tyr取代两个Trp残基表明ZM241385影响了TM6中Trp2466.48和TM7细胞外表面的Trp2466.48的环境和迁移率，导致过渡到疏水性更高的环境。荧光部分IAEDANS被定点引入TM6的细胞内末端（残基2316.33）以报告A2AR的动态细胞质表面。当IAEDANS移至更疏水的环境时，反向激动剂ZM241385引起荧光发射的浓度依赖性增加，这与闭合G蛋白结合缝隙一致。NECA仅产生ZM241385效果的30％。这项研究提供了对SMALP环境的见解；