Measurement of molecular interactions in living cells by fluorescence resonance energy transfer between variants of the green fluorescent protein

Abstract

Many signal transduction pathways operate through oligomerization of proteins into multi-subunit complexes. Although biochemical assays can identify potential protein-protein interactions, studying these interactions in living cells is more challenging. Fluorescence resonance energy transfer (FRET) has been used as a "spectroscopic ruler" to measure molecular proximity, but these methods have been limited by the need for chemical labeling of target proteins or labeled antibodies. We present methods for examining interactions between target proteins molecularly fused to cyan and yellow variants of the green fluorescent protein (GFP) by FRET in living cells. Flow cytometric and microscope-based methods are described that have been applied to a variety of interacting proteins.