Reversible Protein Labeling via Genetically Encoded Dithiolane-Containing Amino Acid and Organoarsenic Probes

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Biology

Background:

• Conventional protein labeling often uses irreversible covalent bonds, restricting dynamic control over modifications.
• Existing methods lack efficient reversibility and can exhibit toxicity.
• There is a need for adaptable tools for real-time protein manipulation in biological systems.

Purpose of the Study:

• To introduce a novel reversible protein labeling strategy.
• To utilize genetically encoded dithiolane-containing amino acid (dtF) and organoarsenic chemistry.
• To demonstrate the utility of this method for dynamic protein modifications and live-cell imaging.

Main Methods:

• Genetically encoded incorporation of a dithiolane-containing amino acid (dtF) into proteins.
• Application of dithiarsolane dicarboxylic acid probe (A2) for protein conjugation.
• Ethanedithiol-mediated removal of the label.
• Assessment of probe toxicity (IC50) and cytotoxicity in live cells.
• Validation in purified proteins (sfGFP-Y151dtF, MYO-K99dtF) and live *Escherichia coli*.

Main Results:

• Near-quantitative protein labeling achieved using probe A2.
• Efficient label removal within 1 hour at room temperature via ethanedithiol.
• Probe A2 demonstrated significantly reduced toxicity compared to arsenoxide (7-fold higher IC50).
• Fluorescent A2-FB derivative showed no cytotoxicity up to 100 μM, suitable for live-cell studies.
• Successful demonstration of dithiol-arsenic chemistry at a single amino acid residue.

Conclusions:

• The developed dithiol-arsenic chemistry provides a reversible protein labeling alternative to dicysteine motifs.
• This method offers a versatile tool for dynamic protein modifications and molecular tracking in biological systems.
• The reduced toxicity and live-cell compatibility enable advanced applications in cell biology and biochemistry.