title: Enhanced dynamic range in a genetically encoded Ca2+ sensor.
abstract: Genetically encoded fluorescence resonance energy transfer (FRET) indicators are powerful tools for real-time detection of second messenger molecules and activation of signal proteins. However, these fluorescent protein-based sensors typically display marginal FRET efficiency. To improve their FRET efficiency for optical imaging and screening, we developed a number of fluorescent protein mutants based on cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). To improve FRET ratios, which were initially within a narrow dynamic range, we used DNA shuffling to develop a new FRET pair called 3xCFP/Venus. The optimized 3xCFP/Venus pair exhibited higher FRET ratios than CyPet/YPet, which has one of the greatest dynamic ranges of protein-based FRET pairs. We converted this FRET pair to a Ca(2+) FRET indicators using circular permutation Venus (cpVenus) linked with 3xCFP to form 3xCFP/cpVenus, which displayed an ∼11-fold change in dynamic range in response to Ca(2+) binding. The enhanced dynamic range for Ca(2+) concentration detection using 3xCFP/cpVenus was confirmed in PC12 cells using previously established indicators (TN-XXL, ECFP/cpCitrine). To our knowledge, this FRET pair displays the largest dynamic range so far among genetically-encoded sensors, and can be used for sensitive FRET detection.
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Shun
authors.initials: S
authors.lastname: Liu
authors.forename: Jun
authors.initials: J
authors.lastname: He
authors.forename: Honglin
authors.initials: H
authors.lastname: Jin
authors.forename: Fei
authors.initials: F
authors.lastname: Yang
authors.forename: Jinling
authors.initials: J
authors.lastname: Lu
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000032:analysis*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D016716:PC12 Cells; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D002118:Calcium