title: Photoswitchable red fluorescent protein with a large Stokes shift.
abstract: A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Kiryl D
authors.initials: KD
authors.lastname: Piatkevich
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
authors.forename: Brian P
authors.initials: BP
authors.lastname: English
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Malashkevich
authors.affiliation: Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Singer
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Department of Biochemistry and Developmental Biology, Institute of Biomedicine, University of Helsinki, Helsinki 00290, Finland. Electronic address: vladislav.verkhusha@einstein.yu.edu.
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
mesh_terms: D002460:Cell Line; D018360:Crystallography, X-Ray; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein