Evolutionary optimization of fluorescent proteins for intracellular FRET.
Fluorescent proteins that exhibit Forster resonance energy transfer (FRET) have made a strong impact as they enable measurement of molecular-scale distances through changes in fluorescence. FRET-based approaches have enabled otherwise intractable measurements of molecular concentrations, binding interactions and catalytic activity, 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.
Nguyen
Annalee W
AW
Daugherty
Patrick S
PS
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D019143:Evolution, Molecular; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins