title: Construction of a fluorescent biosensor family.
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.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: de Lorimier
authors.forename: J Jeff
authors.initials: JJ
authors.lastname: Smith
authors.forename: Mary A
authors.initials: MA
authors.lastname: Dwyer
authors.forename: Loren L
authors.initials: LL
authors.lastname: Looger
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Sali
authors.forename: Chad D
authors.initials: CD
authors.lastname: Paavola
authors.forename: Shahir S
authors.initials: SS
authors.lastname: Rizk
authors.forename: Shamil
authors.initials: S
authors.lastname: Sadigov
authors.forename: David W
authors.initials: DW
authors.lastname: Conrad
authors.forename: Leslie
authors.initials: L
authors.lastname: Loew
authors.forename: Homme W
authors.initials: HW
authors.lastname: Hellinga
mesh_terms: D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D003001:Cloning, Molecular; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism*; D005798:Genes, Bacterial; D005947:Glucose / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D033902:Periplasmic Binding Proteins; D005947:Glucose