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Keywords:

  • Periplasmic binding proteins;
  • biosensor;
  • conformational change;
  • fluorescence, ratiometry;
  • bioinformatics
  • BP, binding protein;
  • bPBP, bacterial periplasmic binding protein;
  • PCR, polymerase chain reaction;
  • ΔIstd, standard intensity change;
  • ΔR, standard ratiometric change;
  • ΔRmax, maximum value of standard ratiometric change;
  • F, fluorescence intensity;
  • FF, fluorescence intensity in ligand-free state;
  • FB, fluorescence intensity in ligand-saturated state;
  • S, ligand concentration;
  • R, fluorescence ratio;
  • RF, fluorescence ratio in ligand-free state;
  • RB, fluorescence ratio in ligand-saturated state;
  • MOPS, 3-morpholinopropanesulfonic acid;
  • NBD, N,N'-dimethyl-N-(iodoacetyl)N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine;
  • NBDE, N-[2-(iodoacetoxy-ethyl]-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole

Abstract

Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand-mediated hinge-bending motion. Construction of cysteine mutations at these locations then permits site-specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three-dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.