An Original Approach to Measure Ligand/receptor Binding Affinity in Non-purified Samples

Several biochemical and biophysical methods are available to determine dissociation constants between a biological target and its ligands. Most of them require purification, labelling or surface immobilisation. However, these measurements remain challenging concerning membrane proteins because purification requires their extraction from the native lipid environment using different approaches, a process that may impact receptor conformation and functionality. We have developed a novel experimental procedure to determine binding affinities of a ligand to a membrane protein, the dopamine D2 receptor (D2R), directly from cell membrane fragments, using microscale thermophoresis (MST). Two main challenges had to be overcome: to determine the concentration of dopamine D2R in the crude sample; to find ways to minimize or account for non-specific binding of the ligand to cell fragments. Using MST, we were able to determine the D2R concentration in cell membrane fragments to be about 36.8 ± 2.6 pmol/mg. Then titration curves allowed the determination of a KD about 5.3 ± 1.7 nM, that is very close to the reported value. Important details of the experimental procedure are detailed to allow the transposition of this novel method to various membrane proteins.