Fig. 2

FP-based analysis of cellular signaling. The behavior of a molecule in a living cell can be analyzed using fluorescent proteins (FPs). a A subcellular localization (or translocation) of a protein can be visualized by expressing FP-fused protein in cells. b The intermolecular interaction can be optically measured as changes in the FRET efficiency between two fused FPs, respectively, to the two target proteins. c The intramolecular protein conformational change can be detected by measuring the FRET efficiency between two FPs fused to the same target protein. d Measurement of protein kinase activity by a FRET-based sensor. The sensor includes a phosphorylation site specific to the kinase and the phospho-amino acid binding domain as well as two FPs located, respectively, at the N and C termini. Phosphorylation of the substrate region by an endogenous kinase allows it to bind to the phospho-amino acid binding domain within the sensor, and the resulting intramolecular conformational change between two FPs can be detected as an increase in FRET efficiency. Similarly, other post-translational modifications, such as glycosylation, methylation and ubiquitination, can also be visualized by including a specific binding domain for the modified peptide in place of the phospho-amino acid binding domain