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G protein-coupled receptors (GPCRs) transmit signals from drugs across cell membranes, leading to associated physiological effects. To study the structural basis of the transmembrane signalling, in-membrane chemical modification (IMCM) has previously been introduced for F-labelling of GPCRs expressed in Spodoptera frugiperda (Sf9) insect cells. Here, IMCM is used with the A adenosine receptor (A AR) expressed in Pichia pastoris; F-NMR revealed nearly complete solvent protection of the A AR transmembrane domain in the membrane and in 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (LMNG)/cholesteryl hemisuccinate (CHS) micelles, and extensive solvent accessibility for A AR in n-dodecyl β-D-maltoside (DDM)/CHS micelles. No Cys residue dominated non-specific labelling with 2,2,2-trifluoroethanethiol. These observations yield an improved protocol for IMCM F-labelling of GPCRs and new insights into variable solvent accessibility for function-related characterization of GPCRs.
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| http://dx.doi.org/10.1002/1873-3468.14627 | DOI Listing |
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