We monitored protein-ligand and protein-protein interactions by imaging cells expressing fusions of the proteins of interest with fluorescent proteins, using fluorescence resonance energy transfer (FRET), sensitive to distance up to 10 nm and orientation changes of the labels.

We studied the effect of protein concentration within cells on FRET rate in model protein fusions: CFP (donor), citrine (acceptor), joined by linkers of 19/23 aminoacids. The FRET methods were ratioing of acceptor to donor emission (donor excitation) and frequency-domain fluorescence lifetime imaging (FLIM). The FRET rate increased at high protein concentration within transfected cells due to dimer/oligomer formation. The mutation A206K in CFP and citrine (shown to decrease dimerization of YFP in solution) drastically decreased the effect of concentration on FRET rate. We also analysed the feasibility of using CFP (donor) and the tetraCysteine-binding biarsenical fluorochrome FlAsH (acceptor) as FRET pair. CFP was tagged with the tetra-Cys-containing peptide ESSGSFLNCCPGCCMEPGGR and transfected in HeLa cells. Fluorescence in the FRET channel increased over time during FlAsH labeling, as the donor CFP was quenched. From CFP data, FRET efficiency was 0.80-0.90. However, from lifetime data it was 0.18. It seems that some FlAsH binds to the tetra-Cys peptide in a non-fluorescent state or conformation causing CFP quench, independent of FRET.

Estrogen Receptor alpha (hERalpha) belongs to the steroid receptor superfamily. To report protein-ligand interactions, we made probes that generate a fluorescent signal proportional to the concentration of ligands of hERalpha, by fusing the receptor with CFP and citrine.