| Abstract
| - The ubiquitous calcium regulating protein calmodulin (CaM) has been utilized as a model drug targetin the design of a competitive binding fluorescence resonance energy transfer assay for pharmacologicalscreening. The protein was labeled by covalently attaching the thiol-reactive fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) to an engineered C-terminalcysteine residue. Binding of the environmentally sensitive hydrophobic probe 2,6-anilinonaphthalenesulfonate (2,6-ANS) to CaM could be monitored by an increase in the fluorescence emission intensityof the 2,6-ANS. Evidence of fluorescence resonance energy transfer (FRET) from 2,6-ANS (acting asa donor) to MDCC (the acceptor in this system) was also observed; fluorescence emission representativeof MDCC could be seen after samples were excited at a wavelength specific for 2,6-ANS. The FRETsignal was monitored as a function of the concentration of calmodulin antagonists in solution.Calibration curves for both a selection of small molecules and a series of peptides based upon knownCaM-binding domains were obtained using this system. The assay demonstrated dose-dependentantagonism by analytes known to hinder the biological activity of CaM. These data indicate that thepresence of molecules known to bind CaM interfere with the ability of FRET to occur, thus leading toa concentration-dependent decrease of the ratio of acceptor:donor fluorescence emission. This assaycan serve as a general model for the development of other protein binding assays intended to screenfor molecules with preferred binding activity.
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