Measurement and Differentiation of Ligand-Induced Calmodulin Conformations by Dual Polarization Interferometry

• Published in: Analytical chemistry (Washington) Vol. 84; no. 3; pp. 1586 - 1591
• Main Authors: Coan, Kristin E. D., Swann, Marcus J., Ottl, Johannes
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 07.02.2012
• Subjects: Analytical chemistry; Animals; Calcium - metabolism; Calmodulin - chemistry; Calmodulin - metabolism; Cattle; Chemistry; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Exact sciences and technology; Interferometry; Lead; Ligands; Measurement; Molecular weight; Protein Binding; Proteins; R&D; Research & development; Trifluoperazine - chemistry; Ligand; Immobilization; Identification; Conformational changes; Density; Characterization; Molecules; Calcium ion; Lead; Molecular mass; Calmodulin; Interferometry; Drug; Use; AIDS; Concentration; Method; Real time; Protein; Infection; Thickness; Chemistry; Viral disease; Inhibitor; Differentiation; Application; Conformation
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac202844e

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/cm3) whereas TFP induced an increase in both thickness (≤0.05 nm) and density (≤0.01 g/cm3). The layer measurements reported here show how DPI can be used to assess and differentiate ligands with distinct structural modes of action.