Figure 2: Arrestin reforms Meta II-P from OpsP and ATR.
From: Distinct loops in arrestin differentially regulate ligand binding within the GPCR opsin
(a) Arrestin was titrated against 4 μM light-induced Meta II-P (black symbols), 4 μM OpsP+40 μM ATR (white symbols) or 4 μM OpsP (grey symbols) in isotonic buffer, and arrestin binding was measured by pull down. Curves were fit as described in the Methods, and apparent Kd and stoichiometry values are listed in Supplementary Table S1. (b) OpsP (4 μM) and ATR (4 μM) were mixed together with or without arrestin (4 μM) in 50 mM HEPES buffer pH 7 without salt. After equilibration, 20 mM t-bHA was added to remove peripheral Schiff bases, followed by acid denaturation and detergent solubilisation. The resulting absorbance spectra of the samples are shown. Inset—difference spectrum calculated by subtracting the spectrum without arrestin from that with arrestin. (c) The time course of regeneration of OpsP (4 μM) by 11-cis-retinal (20 μM) was followed by monitoring the absorbance at 500 nm (arrestin: 4 μM; ATR: 40 μM; 50 mM HEPES buffer pH 7 without salt, 20 °C).
