Extended Data Fig. 3: Minimal cysteine µOR (µOR∆7) shows minor nonspecific labeling by fluorophore or nitroxide spin label.
From: Ligand efficacy modulates conformational dynamics of the µ-opioid receptor
a, µOR∆7 purified in LMNG detergent shows almost no labeling by maleimide ATTO 488 as compared to µOR∆7 in DDM. 3 biological repeats were examined by UV-Vis spectrophotometry and showed similar labeling behavior. For demonstration, one sample was further examined through the gel. CBB, Coomassie Brilliant Blue staining. For gel source data, see Supplementary Fig. 8. b-c, Structures of iodoacetamide Cy3 (b) and Cy5 (c) that were made in-house using NHS-Cy3 and Cy5 from Lumiprobe. d-e, Structures of maleimide Cy3 (d) and Cy7 (e) that are commercially available from Lumiprobe. f, Labeling reactions of the µOR by cysteine-reactive iodoacetamide dye or maleimide dye. g, FRET efficiencies of Cy3/Cy5 and Cy3/Cy7 pairs as a function of inter-dye distances calculated based on R0 values, 55 Å and 40 Å for Cy3/Cy5 and Cy3/Cy7 pairs, respectively. h, Continuous-wave Electron Paramagnetic Resonance (CW-EPR) spectrum for HO-1427 labeled µOR∆7 (green) shows minimal labeling as compared to the same amount of free HO-1427 (blue) and HO-1427 labeled µOR∆7-R182C/R276C (red). i, Labeling reaction of the µOR by of HO-1427.
