title: Allosteric effects of chromophore interaction with dimeric near-infrared fluorescent proteins engineered from bacterial phytochromes.
abstract: Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Сys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs.
authors.affiliation: Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russian Federation.
authors.forename: Olesya V
authors.initials: OV
authors.lastname: Stepanenko
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Baloban
authors.affiliation: Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russian Federation.
authors.forename: Grigory S
authors.initials: GS
authors.lastname: Bublikov
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Daria M
authors.initials: DM
authors.lastname: Shcherbakova
authors.affiliation: Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russian Federation.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Stepanenko
authors.affiliation: Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russian Federation.|Department of Biophysics, Peter the Great St. Petersburg Polytechnic University, St. Petersburg 194064, Russian Federation.
authors.forename: Konstantin K
authors.initials: KK
authors.lastname: Turoverov
authors.affiliation: Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russian Federation.
authors.forename: Irina M
authors.initials: IM
authors.lastname: Kuznetsova
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.|Department of Biochemistry and Developmental Biology, Faculty of Medicine, University of Helsinki, Helsinki 00290, Finland.
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001664:Biliverdine / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008856:Microscopy, Fluorescence; D010834:Phytochrome / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D055550:Protein Stability
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D010834:Phytochrome; D001664:Biliverdine