In‐Membrane Chemical Modification (IMCM) for Site‐Specific Chromophore Labeling of GPCRs

Abstract

We present in-membrane chemical modification (IMCM) for obtaining selective chromophore labeling of intracellular surface cysteines in G-protein-coupled receptors (GPCRs) with minimal mutagenesis.T his method takes advantage of the natural protection of most cysteines by the membrane environment. Practical use of IMCM is illustrated with the site-specific introduction of chromophores for NMR and fluorescence spectroscopyinthe human k-opioid receptor (KOR) and the human A 2A adenosine receptor (A 2A AR). IMCM is applicable to aw ide range of in vitro studies of GPCRs,including single-molecule spectroscopy, and is apromising platform for in-cell spectroscopyexperiments.

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

“…S1A). To achieve specific labeling of the two genetically introduced cysteines, but spare the transmembrane native cysteines, we labeled the receptors in isolated cell membranes, as described previously 49 . After labeling, the receptors were purified and reconstituted in MSP1D1 nanodiscs, which can accommodate only a single monomeric receptor per nanodisc 50 .…”

“…(2) To minimize the effect of fluorophores on the receptor’s dynamics we used small organic dyes attached to strategically engineered cysteines. (3) We avoided the need to remove native cysteines and associated to that potential structural perturbations by using the previously developed in-membrane labeling procedure 49 . We showed that the mutant form of the receptor retains functional activity using the thermal shift assay and cAMP signaling assay in live HEK293T cells.…”

Production of a Human Histamine Receptor for NMR Spectroscopy in Aqueous Solutions

“…19 F-labeled H 1 R was prepared using the in-membrane chemical modification protocol [ 25 ]. Subsequent purification steps followed the above described protocol used for producing u- 15 N H 1 R.…”

“…Samples were concentrated to 280 µL in a Vivaspin-6 concentrator with a 30 kDa MWCO (Sartorius, Goettingen, Germany), 20 µL D 2 O was added and the sample was transferred to 5 mm Shigemi NMR tube. 19 F-labeled H 1 R was prepared using the in-membrane chemical modification protocol [25]. Subsequent purification steps followed the above described protocol used for producing u- 15 N H 1 R.…”

“…These cysteines are C221 5.69 , located at the intracellular end of helix V, cysteine 441 6.61 , located at the extracellular end of helix VI, and C471 7.56 , located at the intracellular end of helix VII (Figure 8a). While the TROSY experiments were recorded at 42 • C, the 19 F NMR data were measured at 7 • C so that we could assess the data in the larger context of many previously reported 19 F NMR GPCR studies [21,25,29,32,33]. Both 19 F spectra of the complexes with doxepin and histamine show complicated line shapes containing more than three individual components (Figure 8b), suggesting the presence of multiple, simultaneously observed conformers for one or more of the labeled cysteines.…”