title: Sterically locked synthetic bilin derivatives and phytochrome Agp1 from Agrobacterium tumefaciens form photoinsensitive Pr- and Pfr-like adducts.
abstract: Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15=C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15=C16 double bond is in either the Z or E configuration and the C14-C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15=C16 Z configuration and a C14-C15 anti conformation in the Pr form and a C15=C16 E configuration and a C14-C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.
authors.affiliation: Division of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan. inomata@cacheibm.s.kanazawa-u.ac.jp
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Inomata
authors.forename: Mostafa A S
authors.initials: MA
authors.lastname: Hammam
authors.forename: Hideki
authors.initials: H
authors.lastname: Kinoshita
authors.forename: Yasue
authors.initials: Y
authors.lastname: Murata
authors.forename: Htoi
authors.initials: H
authors.lastname: Khawn
authors.forename: Steffi
authors.initials: S
authors.lastname: Noack
authors.forename: Norbert
authors.initials: N
authors.lastname: Michael
authors.forename: Tilman
authors.initials: T
authors.lastname: Lamparter
mesh_terms: D016960:Agrobacterium tumefaciens / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry*; D001654:Bile Pigments / Q000737:chemistry*; D001664:Biliverdine / Q000737:chemistry*; D002244:Carbon / Q000737:chemistry; D002845:Chromatography; D004926:Escherichia coli / Q000378:metabolism; D000071677:Histidine Kinase; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry*; D008956:Models, Chemical; D008968:Molecular Conformation; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate; D013053:Spectrophotometry; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; D001654:Bile Pigments; D045342:Light-Harvesting Protein Complexes; D012967:Sodium Dodecyl Sulfate; D002244:Carbon; D011494:Protein Kinases; D000071677:Histidine Kinase; D001664:Biliverdine