Solvent accessibility of a GPCR transmembrane domain probed by in‐membrane chemical modification (IMCM)

Abstract

Edited by Christian GriesingerG 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 19 F-labelling of GPCRs expressed in Spodoptera frugiperda (Sf9) insect cells. Here, IMCM is used with the A 2A adenosine receptor (A 2A AR) expressed in Pichia pastoris; 19 F-NMR revealed nearly complete solvent protection of the A 2A AR transmembrane domain in the membrane and in 2,2-didecylpropane-1,3-bis-b-Dmaltopyranoside (LMNG)/cholesteryl hemisuccinate (CHS) micelles, and extensive solvent accessibility for A 2A AR in n-dodecyl b-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 19 F-labelling of GPCRs and new insights into variable solvent accessibility for function-related characterization of GPCRs.

Membrane Mimetic-Dependence of GPCR Energy Landscapes

“…The chemical shift of component P5 was distinct from reported chemical shifts for free TET, which may be present at trace amounts in 19 F-labeled GPCR samples. 57 The linewidth (FWHM) for component P5 for the complex with Regadenoson was 370 Hz at 280 K, comparable to the line widths for the other observed signals, further supporting the conclusion that the signal P5 originates from the receptor rather than free TET.…”