GREEN FLUORESCENT PROTEIN AS A SIGNAL FOR PROTEIN-PROTEIN INTERACTIONS

Green fluorescent protein (GFP) is autofluorescent. This property has made GFP useful in monitoring in vivo activities such as gene expressi on and protein localization. We find that GFP can be used in vitro to reveal and characterize protein-protein interactions. The interaction between the S-peptide and S-protein fragments of ribonuclease A was ch osen as a model system. GFP-tagged S-peptide was produced, and the int eraction of this fusion protein with S-protein was analyzed by two dis tinct methods: fluorescence gel retardation and fluorescence polarizat ion. The fluorescence gel retardation assay is a rapid method to demon strate the existence of a protein-protein interaction and to estimate the dissociation constant (K-d) of the resulting complex. The fluoresc ence polarization assay is an accurate method to evaluate K-d in a spe cified homogeneous solution and can be adapted for the high-throughput screening of protein or peptide libraries. These two methods are powe rful new tools to probe protein-protein interactions.