Zusammenfassung
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.
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