Intramolecular Fluorescent Resonance Energy Transfer (FRET) by BODIPY Chemical Modification of Cysteine-engineered Mutants of Green Fluorescent Protein

Abstract Green Fluorescent Proteins (GFP) bearing an intramolecular BODIPY fluorophore partner were designed by molecular modeling based on GFP’s three-dimensional structure, and prepared by mutating out native Cys-48 found at the GFP surface (replaced by Ala or Ser) and introducing new specific, surface cysteine residues for chemical modification by a thiol-directed fluorescent BODIPY 507/545 label. This gave derivatives which exhibited strong intramolecular fluorescence resonance energy transfer with quenching of the emission peak of GFP at 508 nm and the appearance of a new FRET emission from the conjugated BODIPY 507/545 around 530 nm.