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.

Solid‐State NMR Spectroscopy to Probe Photoactivation in Canonical Phytochromes

“…As we have shown, the 1 H– 13 C MELODI–HETCOR data of Cph1 and oat phyA3 offered an insight into Pr structural heterogeneity at an atomic level, namely two Pr isoforms ( Pr‐I and Pr‐II ) in terms of their hydrogen‐bonding networks and charge distribution patterns around the chromophore (Fig. ).…”

“…It should be noted that the A ‐ring region of the cofactor in both cases is also found to show more than one conformation ( e.g . the ethylidene substituent) as well as the alternative side‐chain conformations adopted by several amino acids in the N‐terminal region . Hence, it is in principal possible that there are more isomers present, if the two regions involved could change independently, an Occam's razor explanation of the data is that two Pr isoforms coexist in the solution.…”

“…In conjunction with selective isotope labeling (i.e. chromophore labeled in total or at selected positions and of the protein at selected amino acid types) and irradiation techniques within the magnet, MAS NMR offers a particularly attractive route to study the photochemical machinery of photoreceptors down to the atomic level (46)(47)(48)(49)61,62).…”

Tyrosine 263 in Cyanobacterial Phytochrome Cph1 Optimizes Photochemistry at the prelumi‐R→lumi‐R Step

“…MAS NMR at T c revealed that the Pr ground state of Cph1 comprises two isoforms, Pr‐I and Pr‐II, the latter being equivalent to the 2VEA crystal structure . A similar dimorphism is seen for oat phyA3 in the Pr state . It should be noted that a potential effect of the transmitter domain is negligible: The C‐terminus has no effect on spectroscopic and photochemical properties of the pigment as compared with Cph1Δ2; full‐length Cph1 was also shown to be heterogeneous .…”

The family of phytochrome-like photoreceptors: diverse, complex and multi-colored, but very useful

“…Given the difficulty to rationalize color-tuning in microbial and animal rhodopsins, which depend on the 'simpler' chromophore retinal, it is not surprising that theoretical approaches such as full-scale quantum chemical and QM/MM calculations on complex bilin chromophores in their protein environment are still in their infancy [49][50][51]. Here, progress is not only impeded by radiation effects on the chromophore during structural analyses [23 ,52], but also by intrinsic conformational heterogeneity of the P r state and [53][54][55][56] and to some extent also the P fr state [57]. The second issue is the chosen mode of downstream signaling, which apparently differs in the family of phytochrome-like photoreceptors (Figure 4c and d).…”