title: Solid-state NMR spectroscopic study of chromophore-protein interactions in the Pr ground state of plant phytochrome A.
abstract: Despite extensive study, the molecular structure of the chromophore-binding pocket of phytochrome A (phyA), the principal photoreceptor controlling photomorphogenesis in plants, has not yet been successfully resolved. Here, we report a series of two-dimensional (2-D) magic-angle spinning solid-state NMR experiments on the recombinant N-terminal, 65-kDa PAS-GAF-PHY light-sensing module of phytochrome A3 from oat (Avena sativa), assembled with uniformly 13C- and 15N-labeled phycocyanobilin (u-[13C,15N]-PCB-As.phyA3). The Pr state of this protein was studied regarding the electronic structure of the chromophore and its interactions with the proximal amino acids. Using 2-D 13C-13C and 1H-15N experiments, a complete set of 13C and 15N assignments for the chromophore were obtained. Also, a large number of 1H-13C distance restraints between the chromophore and its binding pocket were revealed by interfacial heteronuclear correlation spectroscopy. 13C doublings of the chromophore A-ring region and the C-ring carboxylate moiety, together with the observation of two Pr isoforms, Pr-I and Pr-II, demonstrate the local mobility of the chromophore and the plasticity of its protein environment. It appears that the interactions and dynamics in the binding pocket of phyA in the Pr state are remarkably similar to those of cyanobacterial phytochrome (Cph1). The N-terminus of the region modeled (residues 56-66 of phyA) is highly mobile. Differences in the regulatory processes involved in plant and Cph1 phytochromes are discussed.
authors.affiliation: Leids Instituut voor Chemisch Onderzoek, Universiteit Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Chen
authors.initials: C
authors.lastname: Song
authors.forename: Lars-Oliver
authors.initials: LO
authors.lastname: Essen
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Gärtner
authors.forename: Jon
authors.initials: J
authors.lastname: Hughes
authors.forename: Jörg
authors.initials: J
authors.lastname: Matysik
mesh_terms: D018554:Avena / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D008968:Molecular Conformation; D052980:Phycobilins / Q000737:chemistry / Q000378:metabolism*; D010798:Phycocyanin / Q000737:chemistry / Q000378:metabolism*; D051942:Phytochrome A / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D013194:Staining and Labeling; D045725:Tetrapyrroles / Q000737:chemistry / Q000378:metabolism
chemical_list: D052980:Phycobilins; D051942:Phytochrome A; D020033:Protein Isoforms; D045725:Tetrapyrroles; D010798:Phycocyanin; C011069:phycocyanobilin