Biocompatible click chemistry enabled compartment-specific pH measurement inside E. coli

Abstract

Bioorthogonal reactions, especially the Cu(I)-catalyzed azide-alkyne cycloaddition, have revolutionized our ability to label and manipulate biomolecules under living conditions. The cytotoxicity of Cu(I) ions, however, has hindered the application of this reaction in the internal space of living cells. By systematically surveying a panel of Cu(I)-stabilizing ligands in promoting protein labeling within the cytoplasm of E. coli, here we identify a highly efficient and biocompatible catalyst for intracellular modification of proteins by azide-alkyne cycloaddition. This reaction permits us to conjugate an environment-sensitive fluorophore site-specifically onto HdeA, an acid-stress chaperone that adopts pH-dependent conformational changes, in both the periplasm and cytoplasm of E. coli. The resulting protein-fluorophore hybrid pH indicators enable compartment-specific pH measurement to determine the pH gradient across the E. coli cytoplasmic membrane. This construct also allows the measurement of E. coli transmembrane potential, and the determination of the proton motive force across its inner membrane under normal and acid-stress conditions.

The use of unnatural amino acids to study and engineer protein function

“…None of the twenty natural amino acids conferred a similar enhancement, underlining the unique potential of UAAs in protein engineering and directed evolution. Site-specific conjugation of an environmentally sensitive fluorophore to HdeA, an acid-stress chaperone that undergoes pHdependent conformational changes, was used to create a compartment specific pH-sensor in E. coli [48]. Installation of tyrosine analogues in myoglobin (as model of an oxidase) revealed a linear dependence of O 2 reduction activity on the pKa of the phenol ring, thereby improving the enzyme's activity up to twofold [49].…”

Photo-Triggered Click Chemistry for Biological Applications

“…The only disadvantage is that the copper is toxic to certain cells [90]. Despite efforts to make the copper complexes more biocompatible [91, 92], the breakthrough was achieved by the Bertozzi group [93] through harnessing the ring strain present in cyclooctyne to accelerate the reaction. A variety of cyclooctynes and one cycloheptyne have subsequently been reported [94, 95].…”

Click Chemistry in the Development of Contrast Agents for Magnetic Resonance Imaging

“…Novel biocompatible and bioorthogonal variants of click chemistry have been recently developed. 6 However, traditional, copper-catalyzed azide-alkyne cycloaddition (CuAAC) is still the most commonly used click reaction in the synthesis of contrast agents for MRI due to the limited commercial availability and high cost of other recently developed precursors and components for advanced click chemistry. CuAAC components can be synthesized in bulk for semi-commercial production.…”