Fluorescence Resonance Energy Transfer Between Blue-emitting and
Red-shifted Excitation Derivatives of the Green Fluorescent Protein
Mitra R.D., Silva C.M., Youvan D.C.
Palo Alto Institute for Molecular Medicine, Mountain View, CA 94043, USA.
We report fluorescent resonance energy transfer (FRET) between two linked variants of the
green fluorescent protein (GFP). The C terminus of a red-shifted variant of GFP (RSGFP4)
is fused to a flexible polypeptide linker containing a Factor X a protease cleavage site.
The C terminus of this linker is in turn fused to the N terminus of a blue variant of GFP
(BFP5). The gene product has spectral properties that suggest energy transfer is occurring
from BFP5 to RSGFP4. Upon incubation with Factor Xa, the protein is cleaved,
and the two fluorescent proteins dissociate. This is accompanied by a marked decrease in
energy transfer. The RSGFP4::BFP5 fusion protein demonstrates the feasibility of using
FRET between two GFP derivatives as a tool to monitor protein-protein interactions; in
addition, this construct may find applications as an intracellular screen for protease
inhibitors.