Light-induced chromophore activity and signal transduction in phytochromes observed by ¹³ C and ¹⁵ N magic-angle spinning NMR

Abstract

Both thermally stable states of phytochrome, Pr and Pfr, have been studied by 13 C and 15 N cross-polarization (CP) magic-angle spinning (MAS) NMR using cyanobacterial (Cph1) and plant (phyA) phytochrome sensory modules containing uniformly 13 C-and 15 N-labeled bilin chromophores. Two-dimensional homo-and heteronuclear experiments allowed most of the 13 C chemical shifts to be assigned in both states. Chemical shift differences reflect changes of the electronic structure of the cofactor at the atomic level as well as its interactions with the chromophore-binding pocket. The chromophore in cyanobacterial and plant phytochromes shows very similar features in the respective Pr and Pfr states. The data are interpreted in terms of a strengthened hydrogen bond at the ring D carbonyl. The red shift in the Pfr state is explained by the increasing length of the conjugation network beyond ring C including the entire ring D. Enhanced conjugation within the -system stabilizes the more tensed chromophore in the Pfr state. Concomitant changes at the ring C propionate carboxylate and the ring D carbonyl are explained by a loss of hydrogen bonding to Cph1-His-290 and transmittance of conformational changes to the ring C propionate via a water network. These and other conformational changes may lead to modified surface interactions, e.g., along the tongue region contacting the bilin chromophore.photomorphogenesis ͉ photoreceptors ͉ chromophore-protein interaction ͉ phycocyanobilin ͉ solid-state NMR

Two ground state isoforms and a chromophore D -ring photoflip triggering extensive intramolecular changes in a canonical phytochrome

“…Discrepancies between Pr fluorescence excitation and absorbance spectra of Cph1Δ2 have also been noted and associated with Pr heterogeneity (40). Local doubling of 13 C signals was also detected in our previous DARR NMR experiments on Synechocystis Cph1Δ2 and a recombinant N-terminal fragment of phytochrome A oat (65 kDa, phyA65), implying the coexistence of two Pr isoforms in solution (18). Our current NMR data allow for exact characterization of these, here termed Pr-I and Pr-II (Fig.…”

“…Early NMR spectroscopic studies on proteolytic phytochrome fragments (12,13) indicated that this isomerization occurs at the C15═C16 double bond (for numbering, see Fig. 1A), a geometrical change in line with vibrational spectroscopic investigations (14)(15)(16) and results from recent 13 C solid-state NMR (17,18) in which the most significant changes during the light-triggered conversions are confined to rings C and D. Exact geometries of the chromophore in the Pr state have been resolved as periplanar ZZZssa configurations in bacteriophytochromes from Deinococcus radiodurans (19) and Rhodopseudomonas palustris (20) as well as in the more plant-phytochrome-like Cph1 from the cyanobacterium Synechocystis 6803 (21). On the other hand, the crystal structure of the unusual bacteriophytochrome PaBphP Pseudomonas aeruginosa (22) whose ground state is Pfr shows a ZZEssa conformation, consistent with the expected primary photochemistry at the C15═C16 double bond (Fig.…”

“…The sensitivity enhancement of 1 H indirect dimension was achieved through the use of windowed phase-modulated Lee-Goldburg (wPMLG) homonuclear decoupling scheme (26)(27)(28) over the 1 H evolution period (t 1 ), in our case, wPMLG3 þ . Our previous 2D 13 C-13 C dipolar-assisted rotational resonance (DARR) NMR studies on various states of this protein provided full sets of 13 C chemical shifts of the chromophore (18) and hence the basis for the interpretation of the heteronuclear spectra presented in Fig. 2 as well as Figs.…”

Ultrafast excited-state isomerization in phytochrome revealed by femtosecond stimulated Raman spectroscopy

“…The 20-cm Ϫ1 red-shift of the CAC stretch mode in I* implies a charge redistribution between the C and the D rings, thereby leading to distortion at the C 15 AC 16 double bond. It has been shown that the N-protonated C ring in P r is partially neutralized by a shift of electrons from the D ring that lowers the bond order of the C 15 AC 16 and the C 17 AC 18 bonds (22,23). The resulting partial positive charge on the D ring (Fig.…”

“…There are, however, a few notable differences between the Lumi-R and P fr spectra: the 1,560-cm Ϫ1 (in-phase NH rock for C and D rings) band in Lumi-R is blue-shifted by 10 cm Ϫ1 whereas the HOOP mode is blue shifted by 5 cm Ϫ1 compared with the P fr state. These small differences in peak frequency position in P fr and Lumi-R likely arise from changes in H bonding and/or charge redistribution to the bilin C and D rings occurring in the dark steps subsequent to the photoisomerization that are responsible for the pronounced red-shift of the P fr form (1,23).…”