title: Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence.
abstract: Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed. Finding ways to improve NIR FPs is becoming progressively important. By applying rational design and molecular evolution we have engineered R. palustris bacterial phytochrome into a single-domain NIR FP of 19.6 kDa, termed GAF-FP, which is 2-fold and 1.4-fold smaller than bacterial phytochrome-based NIR FPs and GFP-like proteins, respectively. Engineering of GAF-FP involved a substitution of 15% of its amino acids and a deletion of the knot structure. GAF-FP covalently binds two tetrapyrrole chromophores, biliverdin (BV) and phycocyanobilin (PCB). With the BV chromophore GAF-FP absorbs at 635 nm and fluoresces at 670 nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625 nm and fluoresces at 657 nm. The GAF-FP structure has a high tolerance to small peptide insertions. The small size of GAF-FP and its additional absorbance band in the violet range has allowed for designing a chimeric protein with Renilla luciferase. The chimera exhibits efficient non-radiative energy transfer from luciferase to GAF-FP, resulting in NIR bioluminescence. This study opens the way for engineering of small NIR FPs and NIR luciferases from bacterial phytochromes.
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.|Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia.
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Rumyantsev
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Daria M
authors.initials: DM
authors.lastname: Shcherbakova
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Natalia I
authors.initials: NI
authors.lastname: Zakharova
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Emelyanov
authors.affiliation: Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia.|Department of Biophysics, Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia.
authors.forename: Konstantin K
authors.initials: KK
authors.lastname: Turoverov
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 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: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001664:Biliverdine / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016296:Mutagenesis; D052980:Phycobilins / Q000737:chemistry / Q000378:metabolism; D010798:Phycocyanin / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012241:Rhodopseudomonas / Q000378:metabolism*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D019265:Spectroscopy, Near-Infrared
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D052980:Phycobilins; D010798:Phycocyanin; C011069:phycocyanobilin; D001664:Biliverdine