title: Green fluorescent protein with anionic tryptophan-based chromophore and long fluorescence lifetime.
abstract: Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP-the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: Karen S
authors.initials: KS
authors.lastname: Sarkisyan
authors.affiliation: A.N. Bach Institute of Biochemistry, Moscow, Russia.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Goryashchenko
authors.affiliation: Institute of Gene Biology, Moscow, Russia; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California.
authors.forename: Peter V
authors.initials: PV
authors.lastname: Lidsky
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Gorbachev
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: Nina G
authors.initials: NG
authors.lastname: Bozhanova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Branch, Pushchino, Russia.
authors.forename: Andrey Yu
authors.initials: AY
authors.lastname: Gorokhovatsky
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: Alina R
authors.initials: AR
authors.lastname: Pereverzeva
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: Alina P
authors.initials: AP
authors.lastname: Ryumina
authors.affiliation: A.N. Bach Institute of Biochemistry, Moscow, Russia; Russian Medical Academy of Postgraduate Education, Moscow, Russia.
authors.forename: Victoria V
authors.initials: VV
authors.lastname: Zherdeva
authors.affiliation: A.N. Bach Institute of Biochemistry, Moscow, Russia.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Savitsky
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia.
authors.forename: Kyril M
authors.initials: KM
authors.lastname: Solntsev
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia; Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia; School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia.
authors.forename: Andreas S
authors.initials: AS
authors.lastname: Bommarius
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; Faculty of Medicine, Moscow State University, Moscow, Russia.
authors.forename: George V
authors.initials: GV
authors.lastname: Sharonov
authors.affiliation: Department of Physics, Montana State University, Bozeman, Montana.
authors.forename: Jake R
authors.initials: JR
authors.lastname: Lindquist
authors.affiliation: Department of Physics, Montana State University, Bozeman, Montana.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Drobizhev
authors.affiliation: Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Hughes
authors.affiliation: Department of Physics, Montana State University, Bozeman, Montana; National Insitute of Chemical Physics and Biophysics, Tallinn, Estonia.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Rebane
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Lukyanov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia. Electronic address: mishin@ibch.ru.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Mishin
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000838:Anions / Q000737:chemistry; D004330:Drosophila; D004926:Escherichia coli; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal; D009154:Mutation; D055668:Photochemical Processes; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
chemical_list: D000838:Anions; D049452:Green Fluorescent Proteins; D014364:Tryptophan