Evolutionary optimization of fluorescent proteins for intracellular FRET

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.