title: The N-B Interaction through a Water Bridge: Understanding the Chemoselectivity of a Fluorescent Protein Based Probe for Peroxynitrite.
abstract: Boronic acid and esters have been extensively utilized for molecular recognition and chemical sensing. We recently reported a genetically encoded peroxynitrite (ONOO(-))-specific fluorescent sensor, pnGFP, based on the incorporation of a boronic acid moiety into a circularly permuted green fluorescent protein (cpGFP) followed by directed protein evolution. Different from typical arylboronic acids and esters, the chromophore of pnGFP is unreactive to millimolar concentrations of hydrogen peroxide (H2O2). The focus of this study is to explore the mechanism for the observed unusual chemoselectivity of pnGFP toward peroxynitrite over hydrogen peroxide by using site-directed mutagenesis, X-ray crystallography, (11)B NMR, and computational analysis. Our data collectively support that a His residue on the protein scaffold polarizes a water molecule to induce the formation of an sp(3)-hybridized boron in the chromophore, thereby tuning the reactivity of pnGFP with various reactive oxygen and nitrogen species (ROS/RNS). Our study demonstrates the first example of tunable boron chemistry in a folded nonnative protein, which offers wide implications in designing selective chemical probes.
authors.affiliation: Department of Chemistry, University of California at Riverside , 501 Big Springs Road, Riverside, California 92521, United States.
authors.forename: Zhi-jie
authors.initials: ZJ
authors.lastname: Chen
authors.affiliation: Department of Chemistry, University of California at Riverside , 501 Big Springs Road, Riverside, California 92521, United States.
authors.forename: Ziqi
authors.initials: Z
authors.lastname: Tian
authors.affiliation: Department of Physics and Institute of Molecular Biology, University of Oregon , Eugene, Oregon 97403, United States.
authors.forename: Karen
authors.initials: K
authors.lastname: Kallio
authors.affiliation: Department of Physics and Institute of Molecular Biology, University of Oregon , Eugene, Oregon 97403, United States.
authors.forename: April L
authors.initials: AL
authors.lastname: Oleson
authors.affiliation: Department of Chemistry, University of California at Riverside , 501 Big Springs Road, Riverside, California 92521, United States.
authors.forename: Ao
authors.initials: A
authors.lastname: Ji
authors.affiliation: Department of Chemistry, University of California at Riverside , 501 Big Springs Road, Riverside, California 92521, United States.
authors.forename: Dan
authors.initials: D
authors.lastname: Borchardt
authors.affiliation: Department of Chemistry, University of California at Riverside , 501 Big Springs Road, Riverside, California 92521, United States.
authors.forename: De-en
authors.initials: DE
authors.lastname: Jiang
authors.affiliation: Department of Physics and Institute of Molecular Biology, University of Oregon , Eugene, Oregon 97403, United States.
authors.forename: S James
authors.initials: SJ
authors.lastname: Remington
authors.affiliation: Department of Chemistry, University of California at Riverside , 501 Big Springs Road, Riverside, California 92521, United States.
authors.forename: Hui-wang
authors.initials: HW
authors.lastname: Ai
mesh_terms: D000595:Amino Acid Sequence; D001895:Boron / Q000737:chemistry*; D001896:Boron Compounds / Q000737:chemistry*; D018360:Crystallography, X-Ray; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D006861:Hydrogen Peroxide / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D030421:Peroxynitrous Acid / Q000032:analysis / Q000737:chemistry*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D014867:Water / Q000737:chemistry*
chemical_list: D001896:Boron Compounds; D005456:Fluorescent Dyes; D014867:Water; D030421:Peroxynitrous Acid; D049452:Green Fluorescent Proteins; D010649:Phenylalanine; D006861:Hydrogen Peroxide; D001895:Boron; C033685:4-boronophenylalanine