Bipartite Tetracysteine Display Requires Site Flexibility for ReAsH Coordination

Fluorescence is an essential tool for monitoring molecular interactions, cellular localization and pathway activation both in vitro and in vivo . The development of “pro-fluorescent” bisarsenical dyes in 1998 advanced this field in a significant way, as these dyes provide an alternative labeling strategy that complements some of the limitations of labeling with fluorescent proteins.[ 1 ] These small, cell permeable dyes, 4,5-bis(1,3,2-dithiarsolan-2-yl)fluorescein (FlAsH-EDT 2, Figure S1A ) and 4,5-bis(1,3,2-dithiarsolan-2-yl)resorufin (ReAsH-EDT 2, Figure S1B ) are non-fluorescent in the 1,2-ethanedithiol (EDT) bound form.[ 1 , 2 ] Fluorescence occurs upon thiol-arsenic ligand exchange of EDT for a tetracysteine motif (Cys-Cys-Pro-Gly-Cys-Cys), permitting temporal fluorescent control.[ 1 ] Recently, we reported that the central Pro-Gly in the tetracysteine motif could be replaced with one or more folded proteins domains without a significant loss in bisarsenical affinity or brightness; we refer to this technique as ‘bipartite tetracysteine display’.[ 3 ] We showed, using a set of well characterized model systems, that bipartite tetracysteine display supports fluorescence-based differentiation between folded and unfolded protein states and can detect protein-protein interactions in live cells. The model constructs evaluated in our initial report shared two features in common: the dicysteine motifs were located near the protein N- and/or C-termini and the structures were primarily α-helical. Here we ask whether bipartite display also functions effectively within the context of two more complex protein architectures, p53 and EmGFP, in which the requisite Cys 4 motif is introduced into loops and β-strands.