Title
Enhanced dynamic range in a genetically encoded Ca2+sensor
Author
Shun Liu, Jun He, Honglin Jin, Fei Yang, Jinling Lu, Jie Yang
Year
2011
Journal
Biochemical and Biophysical Research Communications
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 Ca2+ 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 Ca2+ binding. The enhanced dynamic range for Ca2+ 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.
Instrument
FP-6500
Keywords
Fluorescence, FRET, Sensors, Materials, Biochemistry