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Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA
1 Astrobiology Technology Branch, NASA Ames Research Center, Moffett Field, California 94035, USA
2 Center for Biomedical Imaging Technology and Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA
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.
Keywords: Periplasmic binding proteins; biosensor; conformational change; fluorescence, ratiometry; bioinformatics
Abbreviations: BP, binding protein • bPBP, bacterial periplasmic binding protein • PCR, polymerase chain reaction • 
Istd, standard intensity change • R, standard ratiometric change • Rmax, 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
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