title: Biocompatible click chemistry enabled compartment-specific pH measurement inside E. coli.
abstract: Bioorthogonal reactions, especially the Cu(I)-catalysed 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 labelling within the cytoplasm of Escherichia coli, 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.
authors.affiliation: Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Maiyun
authors.initials: M
authors.lastname: Yang
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Abubakar S
authors.initials: AS
authors.lastname: Jalloh
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Institute of Chemistry and BioMedical Sciences, Nanjing University, Nanjing 210093, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Institute of Chemistry and BioMedical Sciences, Nanjing University, Nanjing 210093, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Wu
authors.affiliation: 1] Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China [2] Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
authors.forename: Peng R
authors.initials: PR
authors.lastname: Chen
mesh_terms: D001672:Biocompatible Materials / Q000737:chemistry; D057930:Click Chemistry / Q000379:methods*; D003300:Copper / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004563:Electrochemistry; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008024:Ligands; D008564:Membrane Potentials; D019897:Periplasm / Q000737:chemistry; D010957:Plasmids; D018892:Proton-Motive Force; D017382:Reactive Oxygen Species / Q000737:chemistry
chemical_list: D001672:Biocompatible Materials; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D007477:Ions; D008024:Ligands; D017382:Reactive Oxygen Species; C084501:hdeA protein, E coli; D049452:Green Fluorescent Proteins; D003300:Copper