Exploration of Biarsenical Chemistry—Challenges in Protein Research

Abstract

The fluorescent modification of proteins (with genetically encoded low-molecular-mass fluorophores, affinity probes, or other chemically active species) is extraordinarily useful for monitoring and controlling protein functions in vitro, as well as in cell cultures and tissues. The large sizes of some fluorescent tags, such as fluorescent proteins, often perturb normal activity and localization of the protein of interest, as well as other effects. Of the many fluorescent-labeling strategies applied to in vitro and in vivo studies, one is very promising. This requires a very short (6- to 12-residue), appropriately spaced, tetracysteine sequence (-CCXXCC-); this is either placed at a protein terminus, within flexible loops, or incorporated into secondary structure elements. Proteins that contain the tetracysteine motif become highly fluorescent upon labeling with a nonluminescent biarsenical probe, and form very stable covalent complexes. We focus on the development, growth, and multiple applications of this protein research methodology, both in vitro and in vivo. Its application is not limited to intact-cell protein visualization; it has tremendous potential in other protein research disciplines, such as protein purification and activity control, electron microscopy imaging of cells or tissue, protein-protein interaction studies, protein stability, and aggregation studies.

Specific Inhibition of Sensitized Protein Tyrosine Phosphatase 1B (PTP1B) with a Biarsenical Probe

“…However, the literature precedents concerning the biarsenical cell permeability (or lack thereof) with E. coli , are somewhat mixed. 3 Some authors report that bacterial cell walls are refractory to biarsenicals, 37 whereas some have presented fluorescence data derived from the use of biarsenicals in living E. coli cells. 36 (Perhaps these apparent conflicts arise from differing definitions of “permeable.” Cellular compound concentrations that are sufficient for labeling at the lower limit of fluorescence detection may differ from those needed for full labeling of a target protein.)…”

Bistetrazine‐Cyanines as Double‐Clicking Fluorogenic Two‐Point Binder or Crosslinker Probes

“…Such fluorogenic, bis-quenched two-point bindersa nd cross-linkers exist in the literature; for example, biarsenicals, [30][31][32][33][34][35][36] bisboronic [37] acids or bismaleimide [19,29] probes can be used in tagging schemes in www.chemeurj.org combination with fusion tags involving natural amino acids in an on-natural arrangement (e.g.,C ys 4 or Ser 4 ). Such fluorogenic, bis-quenched two-point bindersa nd cross-linkers exist in the literature; for example, biarsenicals, [30][31][32][33][34][35][36] bisboronic [37] acids or bismaleimide [19,29] probes can be used in tagging schemes in www.chemeurj.org combination with fusion tags involving natural amino acids in an on-natural arrangement (e.g.,C ys 4 or Ser 4 ).…”

PDZ domain from Dishevelled -- a specificity study.

“…This labeling method has many advantages comparing to single cysteine residue labeling with a typical fluorescent sensor. One of them is selective recognition of the tetracysteine sequence and being nonluminescent when a single residue or other cysteine sequences are present (Pomorski & Krężel, 2011). The sterically stable hairpin complex can easily be used for FRET studies (Spagnuolo et al, 2006;Madani et al, 2009).…”