title: Circular permutation of ligand-binding module improves dynamic range of genetically encoded FRET-based nanosensor.
abstract: Quantitative measurement of small molecules with high spatiotemporal resolution provides a solid basis for correct understanding and accurate modeling of metabolic regulation. A promising approach toward this goal is the FLIP (fluorescent indicator protein) nanosensor based on bacterial periplasmic binding proteins (PBPs) and fluorescence resonance energy transfer (FRET) between the yellow and cyan variants of green fluorescent protein (GFP). Each FLIP has a PBP module that specifically binds its ligand to induce a conformation change, leading to a change in FRET between the two GFP variant modules attached to the N- and C-termini of the PBP. The larger is the dynamic range the more reliable is the measurement. Thus, we attempted to expand the dynamic range of FLIP by introducing a circular permutation with a hinge loop deletion to the PBP module. All the six circularly permutated PBPs tested, including structurally distinct Type I and Type II PBPs, showed larger dynamic ranges than their respective native forms when used for FLIP. Notably, the circular permutation made three PBPs, which totally failed to show FRET change when used as their native forms, fully capable of functioning as a ligand binding module of FLIP. These FLIPs were successfully used for the determination of amino acid concentration in complex solutions as well as real-time measurement of amino acid influx in living yeast cells. Thus, the circular permutation strategy would not only improve the performance of each nanosensor but also expand the repertoire of metabolites that can be measured by the FLIP nanosensor technology.
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8561, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Okada
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Ota
authors.forename: Takashi
authors.initials: T
authors.lastname: Ito
mesh_terms: D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism; D001692:Biological Transport; D015374:Biosensing Techniques / Q000379:methods*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000166:cytology
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D008024:Ligands; D033902:Periplasmic Binding Proteins; D049452:Green Fluorescent Proteins