¹⁵N MAS NMR Studies of Cph1 Phytochrome:  Chromophore Dynamics and Intramolecular Signal Transduction

Abstract

Solid-state nuclear magnetic resonance (NMR) is applied for the first time to the photoreceptor phytochrome. The two stable states, Pr and Pfr, of the 59-kDa N-terminal module of the cyanobacterial phytochrome Cph1 from Synechocystis sp. PCC 6803 containing a uniformly 15N-labeled phycocyanobilin cofactor are explored by 15N cross-polarization (CP) magic-angle spinning (MAS) NMR. As recently shown by 15N solution-state NMR using chemical shifts [Strauss, H. M.; Hughes, J.; Schmieder, P. Biochemistry 2005, 44, 8244], all four nitrogens are protonated in both states. CP/MAS NMR provides two additional independent lines of evidence for the protonation of the nitrogens. Apparent loss of mobility during photoactivation, indicated by the decrease of line width, demonstrates strong tension of the entire chromophore in the Pfr state, which is in clear contrast to a more relaxed Pr state. The outer rings (A and D) of the chromophore are significantly affected by the phototransformation, as indicated by both change of chemical shift and line width. On the other hand, on the inner rings (B and C) only minor changes of chemical shifts are detected, providing evidence for a conserved environment during phototransformation. In a mechanical model, the phototransformation is understood in terms of rotations between the A-B and C-D methine bridges, allowing for intramolecular signal transduction to the protein surface by a unit composed of the central rings B and C and its tightly linked protein surroundings during the highly energetic Pfr state.

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

“…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.…”

Characterization of Two Thermostable Cyanobacterial Phytochromes Reveals Global Movements in the Chromophore-binding Domain during Photoconversion

“…That these peaks were below 180 ppm was consistent with all of these nitrogens being protonated at neutral pH (65,66). Interestingly, for Syn-Cph1(PAS-GAF-PHY) two of these peaks overlapped, indicative of similar chemical environments (32,33), whereas in SyB-Cph1(GAF) they were more uniformly resolved, indicative of more distinct chemical environments (Fig. 8B).…”

“…If we assume that the B-and C-rings are in similar chemical environments and are more rigidly held via their propionate side chains (10,19), then the moving pyrrole ring is best assigned to the A-ring. In support, Rohmer et al (33) tentatively assigned a similar 15 N chemical shift, which moved from 146.8 to 142.8 ppm during photoconversion, to the A-ring nitrogen of Synechocystis Cph1(PAS-GAF-PHY) fragment.…”

“…Prior one-dimensional 15 N NMR analysis of Syn-Cph1(PAS-GAF-PHY) assembled with [ 13 C- 15 N]PCB in vitro detected all four pyrrole ring nitrogens in the Pr conformer, with three new distinct peaks appearing after red light irradiation (32,33). These spectra implied that at least three of the chromophore (Fig.…”

“…33). The absence of additional H-N cross-peaks in our 1 H-15 N HSQC Pr/Pfr spectra overlay further implies that the amide of this ring does not change upon photoconversion (see Fig.…”