Chemical reactivity is essential for functional modification of biomolecules with small molecules and the development of covalent drugs. The reactivity between a chemical functional group of a small molecule and that of a large biomolecule cannot be reliably predicted from the reactivity of the corresponding functional groups separately installed on two small molecules, because the proximity effect on reactivity resulting from the binding of the small molecule to the biomolecule is challenging to achieve by mixing two small molecules. Here we present a new strategy to determine the chemical reactivity of two functional groups in the context of close proximity afforded by proteins. The functional groups to be tested were separately installed at the interface of two interacting proteins in the format of amino acid side chains via the expansion of the genetic code. Reaction of the two functional groups resulted in covalent cross-linking of interacting proteins, readily detectable by gel electrophoresis. Using this strategy, we evolved new synthetases to genetically encode N^ε-fluoroacetyllysine (FAcK), an isosteric fluorine analogue of acetyllysine. We demonstrated that fluoroacetamide installed on FAcK, previously thought inert to biological functional groups, actually reacted with the thiol group of cysteine when in proximity. This strategy should be valuable for accurately evaluating chemical reactivity of small molecules toward large biomolecules, which will help avoid undesired side reactions of drugs and expand the repertoire of functional groups to covalently target biomolecules.

Acidic vesicles and organelles play fundamental roles in a broad range of cellular events such as endocytosis, lysosomal degradation, synaptic transmission, pathogen fate, and drug delivery. Fluorescent reporters will be invaluable for studying these complex and multifunctional systems with spatiotemporal resolution, yet common fluorescent proteins are generally nonfluorescent at acidic conditions due to the decrease of anionic chromophores upon protonation, but are fluorescent at physiological pH, creating interfering fluorescence from nonvesicle regions. Here we developed a novel acid-brightening fluorescent protein (abFP) that fluoresces strongly at acidic pH but is nonfluorescent at or above neutral pH, boasting a pH profile opposite to that of common fluorescent proteins. Through expansion of the genetic code, we incorporated a quinoline-containing amino acid Qui into the chromophore of EGFP to reverse the chromophore charge. Protonation of Qui rendered a cationic chromophore, which resulted in unique fluorescence increase only at acidic pH in vitro, in E. coli cells, and on the mammalian cell surface. We further demonstrated that abFP-tagged δ opioid receptors were fluorescently imaged in lysosome showing distinct features and without background fluorescence from other cellular regions, whereas EGFP-tagged receptors were invisible in lysosome. This Qui-rendered cationic chromophore strategy may be generally applied to other fluorescent proteins to generate a palette of colors for acidic imaging with minimal background, and these abFPs should facilitate the study of molecules in association with various acidic vesicles and organelles in different cells and model organisms.

Peroxynitrite is a highly reactive oxidant effecting cell signaling and cell death. Here we report a fluorescent protein probe to selectively detect peroxynitrite. A novel unnatural amino acid, thyronine (Thy), was genetically encoded in E. coli and mammalian cells by evolving an orthogonal tRNA^Pyl/ThyRS pair. Incorporation of Thy into the chromophore of sfGFP or cpsGFP afforded a virtually non-fluorescent reporter. Upon treatment with peroxynitrite, Thy was converted into tyrosine via O-dearylation, regenerating GFP fluorescence in a time- and concentration-dependent manner. Genetically encoded thyronine may also be valuable for other redox applications.

Introducing new chemical reactivity into proteins in living cells would endow innovative covalent bonding ability to proteins for research and engineering in vivo. Latent bioreactive unnatural amino acids (Uaas) can be incorporated into proteins to react with target natural amino acid residues via proximity-enabled reactivity. To expand the diversity of proteins amenable to such reactivity in vivo, a chemical functionality that is biocompatible and able to react with multiple natural residues under physiological conditions is highly desirable. Here we report the genetic encoding of fluorosulfate-l-tyrosine (FSY), the first latent bioreactive Uaa that undergoes sulfur-fluoride exchange (SuFEx) on proteins in vivo. FSY was found nontoxic to Escherichia coli and mammalian cells; after being incorporated into proteins, it selectively reacted with proximal lysine, histidine, and tyrosine via SuFEx, generating covalent intraprotein bridge and interprotein cross-link of interacting proteins directly in living cells. The proximity-activatable reactivity, multitargeting ability, and excellent biocompatibility of FSY will be invaluable for covalent manipulation of proteins in vivo. Moreover, genetically encoded FSY hereby empowers general proteins with the next generation of click chemistry, SuFEx, which will afford broad utilities in chemical biology, drug discovery, and biotherapeutics.