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Mol Biol Cell. 2015 Jun 1;26(11):2054-66. doi: 10.1091/mbc.E14-10-1473. Epub 2015 Apr 15.

Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.

Author information

1
Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Renal Division, Brigham and Women's Hospital, Boston, MA 02115.
2
Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH-Allianz, 69120 Heidelberg, Germany.
3
Department of Physics, Montana State University, Bozeman, MT 59717.
4
Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT 59717.
5
Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114 Instituto de Investigação do Medicamento, Faculdade de Farmácia, Universidade de Lisboa, Lisbon 1640-003, Portugal.
6
National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310.
7
Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114 Broad Institute of Harvard and MIT, Cambridge, MA 02142.
8
Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH-Allianz, 69120 Heidelberg, Germany m.knop@zmbh.uni-heidelberg.de jagesh@hms.harvard.edu.
9
Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Renal Division, Brigham and Women's Hospital, Boston, MA 02115 m.knop@zmbh.uni-heidelberg.de jagesh@hms.harvard.edu.

Abstract

We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein-protein interactions. We also use MPE-FCCS to detect drug-protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

PMID:
25877871
PMCID:
PMC4472016
DOI:
10.1091/mbc.E14-10-1473
[Indexed for MEDLINE]
Free PMC Article

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