QUANTIFICATION OF SMALL MOLECULE-PROTEIN INTERACTIONS BY FRET BETWEEN ENDOGENOUS TRYPTOPHAN RESIDUES AND THE PACIFIC BLUE FLUOROPHORE
Molly M. Lee, Kelsey E. Knewtson, Blake R. Peterson.
The University of Kansas, Lawrence, KS.
In early stage drug development, candidates are identified based on many factors, often starting with the affinity of the active compound for their biological target. For some receptor-ligand systems of medical importance, traditional binding assays can be problematic. We are investigating new methods for measuring receptor-ligand binding affinities, especially in systems where traditional binding assays have proven difficult. We hypothesize that these limitations can be overcome through the development of new assays utilizing Förster resonance energy transfer (FRET). We are investigating the utility of FRET between protein tryptophan residues and Pacific Blue as a readout for small molecule-protein interactions. Tryptophan residues are commonly found near ligand binding sites, so this assay could be applicable to a wide range of systems. To create these and related molecular probes, we have developed a new, practical synthesis that allows the preparation of multigram quantities of the Pacific Blue fluorophore. We demonstrate here the utility of this assay using 3 different receptor-ligand pairs. These include streptavidin-biotin as a model system and the known antibody-antigen binding partners, anti c-myc and c-myc tag peptide. Additionally, we describe the application of this assay to interactions of co-activator proteins with nuclear receptors, a superfamily known for its involvement in cancer progression.