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Evolutionary optimization of fluorescent proteins for intracellular FRET

Nature Biotechnology volume 23, pages 355–360 (2005)Cite this article

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.

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Figure 1: Evolution of FRET pair dynamic range (RRC) and brightness.
Figure 2: Flow cytometric detection of apoptotic cells using FRET.

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Acknowledgements

We wish to acknowledge E. Lipman for helpful discussions, A. Mikhailovsky and D. Korystov for lifetime measurement assistance and P. Bessette for critically reading the manuscript. We further acknowledge the generous support of this project by the National Institutes of Health-National Institute of Biomedical Imaging and Bioengineering grant EB-000205 and a National Science Foundation graduate fellowship to A.W.N.

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Authors and Affiliations

  1. Department of Chemical Engineering, University of California, Santa Barbara, 93106-5080, California, USA

    Annalee W Nguyen & Patrick S Daugherty

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  1. Annalee W Nguyen
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  2. Patrick S Daugherty
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Correspondence to Patrick S Daugherty.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Sorting the synthetically shuffled CFP library (Library 4) for high CFP fluorescence and high FRET-on signal. (PDF 1023 kb)

Supplementary Fig. 2

Fluorescence decay data of fluorescent protein variants. (PDF 358 kb)

Supplementary Fig. 3

Locations of amino acid substitutions in the FRET optimized variants CyPet and YPet. (PDF 695 kb)

Supplementary Fig. 4

Native protein electrophoresis of fluorescent protein variants. (PDF 1170 kb)

Supplementary Fig. 5

Concentration dependent FRET between donor and acceptor pairs. (PDF 69 kb)

Supplementary Table 1

Fluorescence lifetime analysis of CFP and YFP variants (DOC 36 kb)

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Nguyen, A., Daugherty, P. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol 23, 355–360 (2005). https://doi.org/10.1038/nbt1066

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