Site-Specific, Orthogonal Labeling of Proteins in Intact Cells with Two Small Biarsenical Fluorophores

Abstract

The fusion of fluorescent proteins to proteins of interest has greatly advanced fluorescence microscopy, but is often limited by their large size. Here, we report site-specific, orthogonal labeling of two cellular proteins in intact cells with two small fluorescent dyes: fluorescein arsenical hairpin binder, FlAsH, and its red analogue, ReAsH, which bind to tetracysteine motifs. Development of a sequential labeling method to two different motifs, CCPGCC and FLNCCPGCCMEP, allowed site-specific labeling with FlAsH and ReAsH, respectively. Using the cell surface receptor for parathyroid hormone and its cytosolic binding protein, beta-arrestin2, we show their selective visualization in intact cells and analyze their interaction by colocalization and fluorescence resonance energy transfer (FRET). We propose that this method may be widely applied to label intracellular proteins and to study their interactions in intact cells with minimal disturbance of their function.

β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle

“…The labelling was done as described 19,34,35 . In brief, cells were washed twice, with Phenol red-free HBSS containing 1.8 g l −1 glucose (Invitrogen) and incubated at 37 °C and 7% CO 2 for 1 h with 1 ml 250 nM FlAsH in HBSS supplemented with 12.5 µM 1,2-ethane dithiol (EDT).…”

Fluorescent labeling of tetracysteine-tagged proteins in intact cells

“…This approach allowed sequential labeling, but it has so far been used only with purified proteins 44 . A very recent addition to these protocols has been the development of a selective labeling strategy that permits the orthogonal labeling of two different tetracysteine motifs with FlAsH and ReAsH 45 . Such selective labeling allows the simultaneous study in intact cells of two proteins of interest, each labeled with its own color, as well as the investigation of their interactions by colocalization and FRET experiments.…”

“…21). Generally, the contrast should be better when using the high-affinity 12-amino-acid motif compared with the 6-amino-acid motif, as the wash buffer includes higher concentrations of the antidote 21,45 . Multiple repeats of the tetracysteine motif resulted in a modest increase in brightness 46 , but the labeling did not achieve the 1:1 stoichiometry seen for single motifs.…”

“…This includes the activation of G protein–coupled receptors 28,29,42 , as well as the activation of PKC pathways, as a cytosolic PKC-FRET probe was phosphorylated normally after FlAsH labeling 41 . GPCRs labeled with FlAsH or ReAsH have been shown to continue to internalize after agonist stimulation 30,45 , indicating that the endocytotic machinery remains functional, and the receptor recruits β-arrestin to the plasma membrane 45 . Similarly, this labeling approach allows the transport of newly synthesized proteins in dendrites 31 , indicating that targeting of proteins remains functional.…”

Advances in chemical labeling of proteins in living cells

“…FlAsH and ReAsH have also been paired with fluorescent proteins, for example, CFP and GFP, respectively to study conformation changes of individual proteins and protein-protein interactions (Bhattacharya et al, 2012; Hoffmann et al, 2005; Inobe et al, 2011; Roberti et al, 2007). FlAsH and ReAsH have also been paired with each other for FRET experiments by exploiting the different binding affinities between two TC motifs to simultaneously label two proteins in the cell (Zurn et al, 2010). FlAsH and ReAsH have been used as efficient photosensitizers to inactivate TC tagged proteins in CALI experiments (Kasprowicz et al, 2008; Marek and Davis, 2002; Tour et al, 2003).…”