Site-Specific and\nFluorescently Enhanced Installation\nof Post-Translational Protein Modifications via Bifunctional Biarsenical\nLinker

To understand how\nparticular post-translational modifications\n(PTMs)\naffect the function of a target protein, it is essential to first\nprepare and investigate the target with the modification at the desired\nposition. This drives the continuous development of site-specific\nprotein modification technologies. Here, we present the chemical synthesis\nand application of the biarsenical linker SrtCrAsH-EDT<sub>2</sub>, which has a dual labeling functionality. This linker, containing\na sortase A recognition motif, can be conjugated with any protein\ncontaining the LPXTG motif at the C terminus, such as ubiquitin and\nthe SUMO tag, and then attached to a protein of interest (POI) containing\na terminal or bipartite (intramolecularly placed) tetracysteine motif.\nThis modification of the POI facilitates the straightforward and rapid\nincorporation of PTMs, which are further highlighted by the fluorescent\nbiarsenical probe. Consequently, this directly correlates proteins’\nphysical properties and cellular roles under various physiological\nconditions or in disease states. The proposed one-pot labeling methodology\ncan be utilized to explore the effects of PTMs on proteins, affecting\ntheir structure, function, localization, and interactions within the\ncellular environment. Understanding these effects is crucial for uncovering\nthe complex mechanisms that regulate cellular function and dysfunction.