Measurement and differentiation of ligand-induced calmodulin conformations by dual polarization interferometry.

In early drug discovery, knowledge about ligand-induced conformational changes and their influence on protein activity greatly aids the identification of lead candidates for medicinal chemistry efforts. Efficiently acquiring such information remains a challenge in the initial stages of lead finding. Here we investigated the application of dual polarization interferometry (DPI) as a method for the real-time characterization of low molecular weight (LMW) ligands that induce conformational changes. As a model system we chose calmodulin (CaM), which undergoes large and distinct structural rearrangements in response to calcium ion and small molecule inhibitors such as trifluoperazine (TFP). We measured concentration-dependent mass, thickness, and density responses of an immobilized CaM protein layer, which correlated directly with binding and conformational events. Calcium ion binding to CaM induced an increase in thickness (≤0.05 nm) and decrease in density (≤-0.03 g/cm(3)) whereas TFP induced an increase in both thickness (≤0.05 nm) and density (≤0.01 g/cm(3)). The layer measurements reported here show how DPI can be used to assess and differentiate ligands with distinct structural modes of action.