title: Ratiometric analyses at critical temperatures can magnify the signal intensity of FRET-based sugar sensors with periplasmic binding proteins.
abstract: Fluorescence resonance energy transfer (FRET)-based sensors transduce ligand recognition into a change in the fluorophore spectrum, as ligand binding alters the distance between and orientation of two fluorescent proteins. Here, we report a dramatic increase in the signal intensity of FRET-based sugar sensors with bacterial periplasmic binding proteins (PBPs) in the binding moiety, by increasing the analysis temperature, usually higher than 50°C. The increased signal intensity results from a sudden decrease in background signal at critical temperatures, while recovering the maximum FRET ratios in the presence of ligands. When tested with a maltose sensor using a maltose-binding protein as the binding moiety, the FRET ratio at the critical temperature, 55°C, was 17-fold higher than at ambient temperatures. Similar effects were observed using analogous sensors for allose, arabinose, and glucose, providing highly dynamic and quantitative ratio changes at the critical temperatures. The proposed mechanism underlying the signal improvement is thermal relaxation of the binding proteins at the critical temperature; this hypothesis was supported by the results of intrinsic tryptophan fluorescence and circular dichroism experiments. In summary, this study shows that the conformational relaxation of proteins under specific conditions can be leveraged for highly sensitive and rapid measurements of ligands using FRET-based sensors.
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea; College of Interdisciplinary & Creative Studies, Konyang University, Nonsan-si 320-711, Korea.
authors.forename: Jongsik
authors.initials: J
authors.lastname: Gam
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea.
authors.forename: Jae-Seok
authors.initials: JS
authors.lastname: Ha
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea.
authors.forename: Haseong
authors.initials: H
authors.lastname: Kim
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea.
authors.forename: Dae-Hee
authors.initials: DH
authors.lastname: Lee
authors.affiliation: College of Pharmacy, Seoul National University, Seoul 151-742, Korea.
authors.forename: Jeeyeon
authors.initials: J
authors.lastname: Lee
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350, Korea. Electronic address: sglee@kribb.re.kr.
authors.forename: Seung-Goo
authors.initials: SG
authors.lastname: Lee
mesh_terms: D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000032:analysis*; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008320:Maltose / Q000032:analysis / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013696:Temperature
chemical_list: D002241:Carbohydrates; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D033902:Periplasmic Binding Proteins; D008320:Maltose
keywords: Conformation relaxation; Critical temperature; Fluorescence resonance energy transfer; Molecular biosensor