摘要
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 recombi- nant N-terminal, 65-kDa PAS-GAF-PHY light-sensing module of phytochrome A3 from oat （Avena sativa）, assembled with uniformly 13C- and lSN-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 1HJSN 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 inter- facial heteronuclear correlation spectroscopy. 13C doublings of the chromophore A-ring region and the C-ring carbox- ylate 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 （Cphl）. The N-terminus of the region modeled （residues 56-66 of phyA） is highly mobile. Differences in the regulatory processes involved in plant and Cphl phytochromes are discussed.
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 recombi- nant N-terminal, 65-kDa PAS-GAF-PHY light-sensing module of phytochrome A3 from oat （Avena sativa）, assembled with uniformly 13C- and lSN-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 1HJSN 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 inter- facial heteronuclear correlation spectroscopy. 13C doublings of the chromophore A-ring region and the C-ring carbox- ylate 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 （Cphl）. The N-terminus of the region modeled （residues 56-66 of phyA） is highly mobile. Differences in the regulatory processes involved in plant and Cphl phytochromes are discussed.
