title: Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics.
abstract: Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.
authors.affiliation: Department of Physics and Astronomy, VU University Amsterdam, Amsterdam 1081 HV, The Netherlands.
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Hontani
authors.affiliation: Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Daria M
authors.initials: DM
authors.lastname: Shcherbakova
authors.affiliation: Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Baloban
authors.affiliation: Department of Physics and Astronomy, VU University Amsterdam, Amsterdam 1081 HV, The Netherlands.
authors.forename: Jingyi
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, 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
authors.affiliation: Department of Physics and Astronomy, VU University Amsterdam, Amsterdam 1081 HV, The Netherlands.
authors.forename: John T M
authors.initials: JT
authors.lastname: Kennis
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001664:Biliverdine / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D005453:Fluorescence; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010834:Phytochrome / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D019265:Spectroscopy, Near-Infrared
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D010834:Phytochrome; D001664:Biliverdine