Figure 7. Cholesterol allostery in A_2AR is likely a result of indirect membrane effects.

(A) The lipid bilayer can be rigidified and thickened upon addition of cholesterol or an increase in lateral pressure. (B) Averaged fluorescence spectra (n = 4) of Laurdan in A_2AR-embedded nanodiscs containing varying concentrations of cholesterol. (C) The emission intensity of Laurdan at 440 nm and 490 nm were used to calculate the generalized polarization values. Data represent mean ± SEM (n = 4, technical triplicates). Astrisks represent statistical significance over both the 0% and the 3% conditions. Statistical significance was determined via one-way ANOVA followed by Tukey’s multiple comparisons test. ** p ≤ 0.01. (D) ¹⁹F NMR spectra of A_2AR, in the absence of ligand or cholesterol, acquired at 1, 200, 1000, and 2000 bar pressures. The key functional states are indicated by gray dashed lines.