Evolutionary optimization of fluorescent proteins for intracellular FRET
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- 6 February 2005
- journal article
- letter
- Published by Springer Nature in Nature Biotechnology
- Vol. 23 (3) , 355-360
- https://doi.org/10.1038/nbt1066
Abstract
Fluorescent proteins that exhibit Förster resonance energy transfer (FRET) have made a strong impact as they enable measurement of molecular-scale distances through changes in fluorescence1. FRET-based approaches have enabled otherwise intractable measurements of molecular concentrations2, binding interactions3 and catalytic activity4, but are limited by the dynamic range and sensitivity of the donor-acceptor pair. To address this problem, we applied a quantitative evolutionary strategy using fluorescence-activated cell sorting to optimize a cyan-yellow fluorescent protein pair for FRET. The resulting pair, CyPet-YPet, exhibited a 20-fold ratiometric FRET signal change, as compared to threefold for the parental pair. The optimized FRET pair enabled high-throughput flow cytometric screening of cells undergoing caspase-3–dependent apoptosis. The CyPet-YPet energy transfer pair provides substantially improved sensitivity and dynamic range for a broad range of molecular imaging and screening applications.Keywords
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