title: Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.
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.
authors: Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Renal Division, Brigham and Women's Hospital, Boston, MA 02115.
authors: Yinghua
authors: Y
authors: Guan
authors: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH-Allianz, 69120 Heidelberg, Germany.
authors: Matthias
authors: M
authors: Meurer
authors: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH-Allianz, 69120 Heidelberg, Germany.
authors: Sarada
authors: S
authors: Raghavan
authors: Department of Physics, Montana State University, Bozeman, MT 59717.
authors: Aleksander
authors: A
authors: Rebane
authors: Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT 59717.
authors: Jake R
authors: JR
authors: Lindquist
authors: 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.
authors: Sofia
authors: S
authors: Santos
authors: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH-Allianz, 69120 Heidelberg, Germany.
authors: Ilia
authors: I
authors: Kats
authors: National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310.
authors: Michael W
authors: MW
authors: Davidson
authors: Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114 Broad Institute of Harvard and MIT, Cambridge, MA 02142.
authors: Ralph
authors: R
authors: Mazitschek
authors: Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT 59717.
authors: Thomas E
authors: TE
authors: Hughes
authors: Department of Physics, Montana State University, Bozeman, MT 59717.
authors: Mikhail
authors: M
authors: Drobizhev
authors: 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.
authors: Michael
authors: M
authors: Knop
authors: 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.
authors: Jagesh V
authors: JV
authors: Shah
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003593:Cytoplasm; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins; D036641:Microscopy, Fluorescence, Multiphoton; D008969:Molecular Sequence Data; D048328:Potoroidae; D011485:Protein Binding; D055503:Protein Multimerization; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence / Q000379:methods*; D000097573:Red Fluorescent Protein
chemical_list: D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C485182:Cyan Fluorescent Protein