Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein

Abstract

Publisher Summary Green fluorescent protein (GFP) is a spontaneously fluorescent protein from the jellyfish Aequorea victoria . It can be genetically concatenated to many other proteins, and the resulting fusion proteins are usually fluorescent and preserve the biochemical functions and cellular localization of the partner proteins. GFP fusions have major advantages for fluorescent labeling of proteins by covalent reaction with small molecule dyes. The chimeric fluorescent proteins are generated in situ by gene transfer into cells or organisms, obviating high-level heterologous expression, purification, in vitro labeling, and microinjection of recombinant proteins. Targeting signals can be used to direct localization of the chimeras to particular tissues, cells, organelles, or subcellular sites. Mutagenesis has produced GFP mutants with shifted wavelengths of excitation and emission that can serve as donors and acceptors for fluorescence resonance energy transfer (FRET). FRET between GFP mutants fused to host proteins has great potential as a general method for imaging dynamic changes in ligand concentrations and the conformations and interactions of those host proteins.