Modification of the zonal elution method for detection of transient protein-protein interactions involving ligand exchange.

A new affinity chromatography method was developed by modifying a zonal elution method. The new method targets transient protein-protein interactions, such as those encountered during direct ligand transfer between the ligand transporter and its cognate receptor. A ligand-loaded transport protein is immobilized on the solid support, and a plug containing a putative receptor is flowed through the column. Elution profiles of proteins not interacting with the immobilized transporter can be approximated with a simple Gaussian curve, while the elution profiles of cognate receptors show significant delay and exhibit complex shape. Ligand transfer from the immobilized transporter molecules to the receptors is verified by both UV absorbance measurements and mass spectrometry. The sensitivity of the method is demonstrated using retinoic acid (RA) transfer from various isoforms of cellular RA binding proteins (CRABPs) and RA receptor γ (RARγ). Although these interactions have been hypothesized long ago to proceed via direct mechanism (i.e., via transient docking of the receptor and the transporter), the existing biophysical techniques failed to detect the presence of the transporter-receptor complexes. However, the modified zonal elution method provides unequivocal evidence of direct interaction between RARγ and one of the CRABP isoforms (CRABP II) during the ligand transfer to the receptor.