DOI: 10.1073/pnas.0509922103. A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP

A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP

10.1073/pnas.0509922103

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

Förster resonance energy transfer (FRET) microscopy is a powerful technique that enables the visualization of signaling intermediates, protein interactions, and protein conformational and biochemical status. With the availability of an ever-increasing collection of fluorescent proteins, pairs of spectrally different variants have been used for the study of FRET in living cells. However, suitable spectral overlap, necessary for efficient FRET, is limited by the requirement for proper emission separation. Currently used FRET pairs represent compromises between these opposing spectral demands that reduce the maximally attainable FRET sensitivity. We present a previously undescribed FRET acceptor, a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein). REACh allows the use of the photophysically superior FRET donor EGFP, with which it exhibits optimal spectral overlap, which obviates the need for narrow spectral filtering and allows additional fluorescent labels to be used within the same cell. The latter allows the generation of sophisticated bioassays for complex biological questions. We show that this dark acceptor is ideally suited for donor fluorescence lifetime imaging microscopy (FLIM) and confirm these measurements with an independent intensity-based donor fluorescence quenching resonance energy transfer (FqRET) assay. REACh also can be used in donor photobleaching kinetics-based FRET studies. By detecting FRET between a GFP-tagged ubiquitination substrate and REACh-labeled ubiquitin, we imaged the active ubiquitination machinery inside cells. This assay therefore can be used to study proteins whose function is regulated by ubiquitination.

Bibliography

Ganesan, S., Ameer-beg, S. M., Ng, T. T. C., Vojnovic, B., & Wouters, F. S. (2006). A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for FÃ¶rster resonance energy transfer with GFP. Proceedings of the National Academy of Sciences, 103(11), 4089â4094.

Authors 5

Sundar Ganesan (first)
Simon M. Ameer-beg (additional)
Tony T. C. Ng (additional)
Borivoj Vojnovic (additional)
Fred S. Wouters (additional)

References 26 Referenced 180

10.1002/andp.19484370105
10.1016/S0962-8924(01)01982-1
10.1016/S0074-7696(04)37005-1
10.1126/stke.2000.38.pl1
10.1146/annurev.biochem.67.1.509
10.1038/nbt945
10.1042/BJ20040321
R. M. Clegg B. A. Feddersen E. Gratton T. M. Jovin 1640 448–460 (1992). (10.1117/12.58237)
10.1046/j.1365-2818.1999.00427.x
10.1364/AO.39.006306
10.1046/j.1365-2818.1998.00351.x
W. Becker A. Bergmann K. Konig U. Tirlapur 4262 414–419 (2001). (10.1117/12.424584)
10.1038/13657
10.1038/nbt1037
10.1073/pnas.0407752101
10.1038/84443
10.1002/jemt.20078
10.1016/S0301-0104(98)00245-6
10.1006/abio.2000.4708
10.1074/jbc.273.52.34970
10.1038/ncb1059
A. G. Harpur, P. I. H. Bastiaens Molecular Cloning: A Laboratory Manual, eds J. Sambrook, D. W. Russel (Cold Spring Harbor Lab. Press, Woodbury, NY) Vol. 3, 18.69–18.95 (2001). / Molecular Cloning: A Laboratory Manual by Harpur A. G. (2001)
10.1016/0030-4018(96)00189-7
10.1042/bj20021191
10.1073/pnas.182157199
10.1016/S0014-5793(01)03263-X

Dates

Type	When
Created	19 years, 5 months ago (March 6, 2006, 8:34 p.m.)
Deposited	7 months, 4 weeks ago (Jan. 7, 2025, 6:03 p.m.)
Indexed	1 week, 4 days ago (Aug. 26, 2025, 2:34 a.m.)
Issued	19 years, 6 months ago (March 6, 2006)
Published	19 years, 6 months ago (March 6, 2006)
Published Online	19 years, 6 months ago (March 6, 2006)
Published Print	19 years, 5 months ago (March 14, 2006)

Funders 0

None

BibTeX

@article{Ganesan_2006, title={A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP}, volume={103}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.0509922103}, DOI={10.1073/pnas.0509922103}, number={11}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Ganesan, Sundar and Ameer-beg, Simon M. and Ng, Tony T. C. and Vojnovic, Borivoj and Wouters, Fred S.}, year={2006}, month=mar, pages={4089–4094} }

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