Conformational mobility of immobilized proteins

Cellular membrane fragments have been immobilized on the surface of a silica-based liquid chromatographic support and on the surface of glass capillaries to create immobilized receptor and drug transporter columns. These columns have included phases containing one subtype of the nicotinic receptor (α3β2, α3β4, α4β2, α4β4) and the P-glycoprotein transporter. A key question in the application of these columns to drug discovery and development is the ability of the immobilized receptor or transporter to undergo ligand and/or co-factor induced conformational changes. Using frontal affinity chromatographic techniques and non-linear chromatographic techniques it has been demonstrated that the immobilized nicotinic receptors undergo agonist-induced conformational shifts from the resting to desensitized states with corresponding changes in binding affinities and enantioselectivities. Ligand-induced allosteric interactions and ATP-driven conformational changes have also been demonstrated with the immobilized Pgp stationary phase. The results demonstrate that the immobilized proteins retained their ability to undergo conformational mobility and that this is an attractive alternative to allow for the full characterization of multiple protein conformations.