EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.

A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at approximately 390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Forster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.

[1]  J. Kyhse-Andersen.  Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. , 1984, Journal of biochemical and biophysical methods.

[2]  M. J. Cormier,et al.  Primary structure of the Aequorea victoria green-fluorescent protein. , 1992, Gene.

[3]  T. Honjo,et al.  The recombination signal sequence-binding protein RBP-2N functions as a transcriptional repressor , 1994, Molecular and cellular biology.

[4]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[5]  S. Boxer,et al.  Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Douglas C. Youvan,et al.  Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions , 1996 .

[7]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[8]  W. Webb,et al.  Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[9]  S. Lukyanov,et al.  Fluorescent proteins from nonbioluminescent Anthozoa species , 1999, Nature Biotechnology.

[10]  Y Chen,et al.  Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far‐red fluorescence , 2000, FEBS letters.

[11]  K. Spindler,et al.  Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria). , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Raphael Kopan,et al.  Notch signaling: from the outside in. , 2000, Developmental biology.

[13]  J Michiels,et al.  Identification of different emitting species in the red fluorescent protein DsRed by means of ensemble and single-molecule spectroscopy , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[14]  S. Boxer,et al.  Photophysics of DsRed, a Red Fluorescent Protein, from the Ensemble to the Single-Molecule Level , 2001 .

[15]  F. Oswald,et al.  p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1 , 2001, Molecular and Cellular Biology.

[16]  M. Falk,et al.  Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed‐tagged proteins1 , 2001, FEBS letters.

[17]  A Miyawaki,et al.  Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer. , 2001, Biochemistry.

[18]  Horst Hameister,et al.  SHARP is a novel component of the Notch/RBP‐Jκ signalling pathway , 2002 .

[19]  George H. Patterson,et al.  A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells , 2002, Science.

[20]  A. Miyawaki,et al.  An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[21]  G Ulrich Nienhaus,et al.  A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria) , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[22]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  S. Lukyanov,et al.  Kindling fluorescent proteins for precise in vivo photolabeling , 2003, Nature Biotechnology.

[24]  A. Miyawaki,et al.  A Green-emitting Fluorescent Protein from Galaxeidae Coral and Its Monomeric Version for Use in Fluorescent Labeling* , 2003, Journal of Biological Chemistry.

[25]  Atsushi Miyawaki,et al.  Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. , 2003, Molecular cell.

[26]  Franz Oswald,et al.  Identification of GFP-like Proteins in Nonbioluminescent, Azooxanthellate Anthozoa Opens New Perspectives for Bioprospecting , 2004, Marine Biotechnology.

[27]  Chung-Ho Sun,et al.  Fluorescence correlation spectroscopy investigation of a GFP mutant-enhanced cyan fluorescent protein and its tubulin fusion in living cells with two-photon excitation. , 2004, Journal of biomedical optics.

[28]  S. Lukyanov,et al.  GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. , 2004, Molecular biology and evolution.

[29]  G Ulrich Nienhaus,et al.  Photodynamics of red fluorescent proteins studied by fluorescence correlation spectroscopy. , 2004, Biophysical journal.