A high-throughput fluorescence chemical denaturation assay as a general screen for protein-ligand binding.
Chemical denaturation of ligand-protein complexes can provide the basis of a label-free binding assay. Here, we show how the technique can be used as a sensitive/affordable screen of potential ligands from a pool of lead drug variants. To demonstrate, we characterized the binding of polyketide ligands based on the mTOR inhibitor rapamycin to the cellular immunophilin FKBP12. This used the intrinsic fluorescence of the protein to monitor the chemical denaturation of each FKBP12-ligand complex. The assay was then successfully modified to a 96-well plate-based screen. Both formats were able to differentiate binding affinities across a wide dynamic range.
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.
Kumaran
K
Mahendrarajah
Paul A
PA
Dalby
Barrie
B
Wilkinson
Sophie E
SE
Jackson
Ewan R G
ER
Main
D005453:Fluorescence; D005470:Fluorometry; D057166:High-Throughput Screening Assays / Q000379:methods*; D008024:Ligands; D011485:Protein Binding; D011489:Protein Denaturation; D020123:Sirolimus / Q000737:chemistry / Q000378:metabolism; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D008024:Ligands; D022061:Tacrolimus Binding Protein 1A; D020123:Sirolimus