Labeling Tetracysteine‐Tagged Proteins with a SplAsH of Color: A Modular Approach to Bis‐Arsenical Fluorophores

Abstract

Everything nicely tied up. We have synthesized a non‐fluorescent bis‐arsenical targeting moiety that can be tethered to a wide variety of different fluorescent payloads. This strategy greatly broadens the scope of dyes that can be used to label tetracysteine‐tagged proteins.

Specific Biarsenical Labeling of Cell Surface Proteins Allows Fluorescent- and Biotin-tagging of Amyloid Precursor Protein and Prion Proteins

“…Because of the potential power of TC-tagging, we and others have developed new biarsenical derivatives (Bhunia and Miller, 2007;Cao et al, 2007;Chen et al, 2007;Liu et al, 2007;Tour et al, 2007). We synthesized FlAsH conjugates containing either an Alexa Fluor dye or biotin that worked as efficiently and specifically as FlAsH, suggesting that conjugation to carboxy-FlAsH would not substantially affect the affinity of the biarsenical moiety for the TC-motif.…”

“…This is important because such probes are useful only when they are specific. Biarsenical scaffolds conjugated to Alexa Fluor dye (Bhunia and Miller, 2007) or biotin (with an additional dopamine moiety) (Liu et al, 2007) have been described by other groups, but the affinity and specificity of these reagents for the minimal CCPGCC TC motif remain to be shown in the context of the highly challenging application of labeling TC-tagged proteins expressed in mammalian cells where the ratio of TC-tagged protein to total protein is usually very low and off-target biarsenical-binding proteins are common (Griffin et al, 2000;Stroffekova et al, 2001). Furthermore, without a method to label cell-surface TCtagged proteins these membrane-impermeable biarsenical compounds would be restricted to cell-free or fixed/permeabilized cell applications.…”

Studying the Dynamics of Flagella in Multicellular Communities of Escherichia coli by Using Biarsenical Dyes

“…The tightly regulated assembly of the flagella, the structure of the FliC protein, and the dimensions of the channel/pore in the filament make the fusion of a fluorescent protein to the N-or C-terminal region of FliC an incompatible approach for engineering fluorescent labels into the flagella (42). In contrast, (1), (ii) the quantum yield increases significantly (ϳ0.5) upon binding to the TC motif (1), (iii) they are synthesized using straightforward chemistry (2), (iv) the label can be removed using millimolar concentrations of 1,2-ethanedithiol (EDT) or 2,3-dimercaptopropanol (1), (v) TC-labeled proteins are more likely to retain their native function during investigations via fluorescent microscopy than fluorescent proteins (31), and (vi) a variety of biarsenical fluorophores that have unique physical and optical properties are available (7,11,12,45,49). Here we demonstrate the application of the biarsenical dyes FlAsH and ReAsH to specifically label the primary flagellar filament protein of E. coli, FliC ( Fig.…”

A Targeted Releasable Affinity Probe (TRAP) for In Vivo Photocrosslinking

“…It is expected that useful results can then be achieved with the existing TRAP crosslinker after reaction scale-up, retagging of the proteins, and expression of RNA polymerase in SlyD deletion mutants. Parallel efforts will aim to enhance crosslinking efficiencies through the synthesis of new targeted reversible crosslinkers based on the multiuse affinity probes AsCy3, [57] ReAsH, [16] as well as perfluorinated, [58] and spirolactam biarsenicals, [59] which due to their shifted fluorescence will be less-efficient quenchers for benzophenone. Azide and nitrobenzene-based crosslinking units will be substituted for benzophenone in an effort to reduce the in vivo photoactivation times.…”