Advances in chemical labeling of proteins in living cells

Abstract

The pursuit of quantitative biological information via imaging requires robust labeling approaches that can be used in multiple applications and with a variety of detectable colors and properties. In addition to conventional fluorescent proteins, chemists and biologists have come together to provide a range of approaches that combine dye chemistry with the convenience of genetic targeting. This hybrid-tagging approach amalgamates the rational design of properties available through synthetic dye chemistry with the robust biological targeting available with genetic encoding. In this review, we discuss the current range of approaches that have been exploited for dye targeting or for targeting and activation and some of the recent applications that are uniquely permitted by these hybrid-tagging approaches.

Figure 1. Chemical labeling of proteins of interest

(a) The protein of interest is fused to a fluorescent protein with intrinsic fluorescence. (b) The protein of interest is fused to a protein domain tag, which can bind to a fluorophore derivative through covalent or non-covalent interactions. (c) The protein of interest is fused to a peptide tag, which then acts as the enzyme substrate for covalent fluorophore attachment. (d) The protein of interest is fused to a protein or peptide tag, which then binds and activates a fluorogen through covalent or non-covalent interactions.

Figure 2

Structure and spectral properties of TO1-CypHer5 (Grover et al., 2012). (a) Structure of TO1-CypHer5. TO1 is linked to pH dependent CypHer5, forming a FRET pair. The pH sensitivity is conferred by the protonation of the free indole nitrogen atom. (b) The emission spectra of TO1-CypHer5 when bound to FAP at various pH, normalized to the TO1 emission peak. Excitation wavelength: 488 nm. (c – c’) Color change of TO1-CypHer5 during FAP tagged β2-AR endocytosis. Upon addition of the agonist, isoproterenol (10 μM) to the cells, FAP-β2-AR undergoes endocytosis, leading to a significant increase of red emission from the acidic vesicles while the plasma membrane (pH 7.4) still shows exclusively green emission. Scalebar 10 μm.