Dark dyes-bright complexes: fluorogenic protein labeling

Abstract

Complexes formed between organic dyes and genetically encoded proteins combine the advantages of stable and tunable fluorescent molecules and targetable, biologically integrated labels. To overcome the challenges imposed by labeling with bright fluorescent dyes, a number of approaches now exploit chemical or environmental changes to control the properties of a bound dye, converting dyes from a weakly fluorescent state to a bright, easily detectable complex. Optimized, such approaches avoid the need for removal of unbound dyes, facilitate rapid and simple assays in cultured cells and enable hybrid labeling to function more robustly in living model organisms.

Figure 1. Spectroscopic changes utilized for fluorogen activation

Binding of a dye to a protein domain can result in changes in quantum yield (top), excitation wavelength (middle) or extinction coefficient/excitation cross-section (bottom). Combinations of these properties will result in high activation ratios.

Figure 2. Fluorogen activation approaches

Binding of the fluorogen to an expressed domain can be used to release a quenching group (top), to bring a dye near to an environment that activates it, for example a membrane or protein pocket (middle), or to directly bind the fluorogen dye in a fluorescent complex (bottom). Direct binding of the fluorogen to the fluorogen activating protein is reversible, unlike cleavage or covalent linkage, which is advantageous for some applications.

Figure 3. Fluorogen dye structures spanning the visible spectral range

Fluorogenic dyes of various structure and spectral properties have been demonstrated for labeling of living cells. The dye structure is shown, along with the activating protein (as named in the cited references), and the reported activation ratio (AR). References: OTB-SO₃ [25]; TMBDMA [28]; YC20 [21]; TO1 [12]; α-CN-DIR [27]; DIR [26]; MG [30]; JF646 [24].

Figure 4. Fluorogen labeling for protein trafficking at the membrane

Pulse-treat-chase labeling of cell-surface receptors with exchangeable fluorogens of two resolvable colors results in differential coding of proteins protected (from exchange) in endosomes and those still exposed at the plasma membrane.