Figure 2
From: Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor
Behavior in solution and stability of purified native A2AR. (A) Gel filtration profile of A2AR showing two peaks (indicated as 1 and 2). Fractions corresponding to each peak were pooled, concentrated and used to run a second SEC as indicated by the red and black chromatograms. (B) Gel filtration fractions were analyzed by SDS-PAGE revealed by stain free (total protein) or western blot (A2AR only). Full length original gels are presented in Fig. S5. SEC-MALS analysis show the profile of peak 1 (C) and peak 2 (E). Light scattering (LS), differential refractive index (dRI), OD at 280 nm (y1 axes) and calculated masses (y2 axes) were plotted as a function of experiment’s time. OD, LS and dRI were rescaled to range from 0 to 100%, with 100% corresponding to maximum value of the curve. Negative stain image of A2AR fractions from Size exclusion chromatography peak 1 (D) and peak 2 (F). Scale bar correspond to 100 nm. Stability of solubilized/purified A2AR by Analytical Size exclusion chromatography (G). SEC was performed on affinity purified A2AR and gel filtrated protein (pool 2) after incubation 1 and 7 days at room temperature. SEC chromatograms were superimposed. Thermalshift assay (H). The assay was performed as described in methods on wild type and full length A2AR solubilized using two different conditions, CALX and reference corresponding to DDM/CHS/CALX-R10/ZM241385 and DDM/CHS, respectively. For comparison, A2AR StaR2, truncated (96 aminoacid c-terminal deletion) and mutated 8-point mutations solubilized and purified in DDM/CHS as described78 was also analyzed by thermalshift.
