Exploring Chromophore-Binding Pocket: High-Resolution Solid-State H-C Interfacial Correlation NMR Spectra with Windowed PMLG Scheme.

High-resolution two-dimensional (2D) (1)H-(13)C heteronuclear correlation spectra are recorded for selective observation of interfacial 3-5.5 Å contacts of the uniformly (13)C-labeled phycocyanobilin (PCB) chromophore with its unlabeled binding pocket. The experiment is based on a medium- and long-distance heteronuclear correlation (MELODI-HETCOR) method. For improving (1)H spectral resolution, a windowed phase-modulated Lee-Goldburg (wPMLG) decoupling scheme is applied during the t(1) evolution period. Our approach allows for identification of chromophore-protein interactions, in particular for elucidation of the hydrogen-bonding networks and charge distributions within the chromophore-binding pocket. The resulting pulse sequence is tested on the cyanobacterial (Cph1) phytochrome sensory module (residues 1-514, Cph1Δ2) containing uniformly (13)C- and (15)N-labeled PCB chromophore (u-[(13)C,(15)N]-PCB-Cph1Δ2) at 17.6 T.

On the collective nature of phytochrome photoactivation.

The red/far-red-sensing biological photoreceptor phytochrome is a paradigmatic two-state signaling system. The two thermally stable states are interconverted via a photoreaction of the covalently bound tetrapyrrole chromophore. Applying recently developed solid-state nuclear magnetic resonance, we study both the chromophore and its protein pocket in the Pr (red-absorbing) and Pfr (far-red-absorbing) states. The observations show that the phototransformation combines local chemical reactions with a mesoscopic transition of order. Both the chromophore and its binding pocket are quasi-liquid and disordered in Pr, yet quasi-solid and ordered in Pfr. Possible biochemical implications are discussed.

Spectroscopy and a high-resolution crystal structure of Tyr263 mutants of cyanobacterial phytochrome Cph1.

Phytochromes are biliprotein photoreceptors that can be photoswitched between red-light-absorbing state (Pr) and far-red-light-absorbing state (Pfr). Although three-dimensional structures of both states have been reported, the photoconversion and intramolecular signaling mechanisms are still unclear. Here, we report UV-Vis absorbance, fluorescence and CD spectroscopy along with various photochemical parameters of the wild type and Y263F, Y263H and Y263S mutants of the Cph1 photosensory module, as well as a 2.0-Å-resolution crystal structure of the Y263F mutant in its Pr ground state. Although Y263 is conserved, we show that the aromatic character but not the hydroxyl group of Y263 is important for Pfr formation. The crystal structure of the Y263F mutant (Protein Data Bank ID: 3ZQ5) reaffirms the ZZZssa chromophore configuration and provides a detailed picture of its binding pocket, particularly conformational heterogeneity around the chromophore. Comparison with other phytochrome structures reveals differences in the relative position of the PHY (phytochrome specific) domain and the interaction of the tongue with the extreme N-terminus. Our data support the notion that native phytochromes in their Pr state are structurally heterogeneous.

Signaling kinetics of cyanobacterial phytochrome Cph1, a light regulated histidine kinase.

Cyanobacterial phytochrome 1 (Cph1) is a red/far-red light regulated histidine kinase, which together with its response regulator (Rcp1) forms a two-component light signaling system in Synechocystis 6803. In the present study we followed the in vitro autophosphorylation of Cph1 and the subsequent phosphotransfer to Rcp1 in different ionic milieus and following different light treatments. Both processes were red/far-red reversible with activity manifested in the Pr ground state (in darkness or after far-red irradiation) and with strongest activities being exhibited in the presence of Mn(2+). In vivo and in vitro assembled holoproteins in the Pr state displayed at least 4-fold higher efficiencies (k(cat)/K(m)) for autophosphorylation and phosphotransfer than the apoprotein or the holoprotein at photoequilibrium in red light. The reduced activities observed following red light treatments were consistent with the Pfr state being enzymatically inactive. Thus, both the rate of kinase autophosphorylation and the rate of phosphotransfer regulate the phosphorylation state of the response regulator, consistent with the rotary switch model regulating accessibility of the histidine target.

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

Phytochrome photoreceptors mediate light responses in plants and in many microorganisms. Here we report studies using (1)H-(13)C magic-angle spinning NMR spectroscopy of the sensor module of cyanobacterial phytochrome Cph1. Two isoforms of the red-light absorbing Pr ground state are identified. Conclusive evidence that photoisomerization occurs at the C15-methine bridge leading to a ß-facial disposition of the ring D is presented. In the far-red-light absorbing Pfr state, strong hydrogen-bonding interactions of the D-ring carbonyl group to Tyr-263 and of N24 to Asp-207 hold the chromophore in a tensed conformation. Signaling is triggered when Asp-207 is released from its salt bridge to Arg-472, probably inducing conformational changes in the tongue region. A second signal route is initiated by partner swapping of the B-ring propionate between Arg-254 and Arg-222.

Spectroscopic and photochemical characterization of the red-light sensitive photosensory module of Cph2 from Synechocystis PCC 6803.

Cyanobacterial phytochromes are a diverse family of light receptors controlling various biological functions including phototaxis. In addition to canonical bona fide phytochromes of the well characterized Cph1/plant-like clade, cyanobacteria also harbor phytochromes that absorb green, violet or blue light. The Synechocystis PCC 6803 Cph2 photoreceptor, a phototaxis inhibitor, is unconventional in bearing two distinct chromophore-binding GAF domains. Whereas the C-terminal GAF domain is most likely involved in blue-light perception, the first two domains correspond to a Cph1-like photosensory module lacking the PAS domain. Biochemical and spectroscopic studies show that this region switches between red (P(r) ) and far-red (P(fr) ) absorbing states. Unlike Cph1, the P(fr) state of Cph2 decays rapidly in darkness. Mutations close to the PCB chromophore further destabilize the P(fr) state without drastically affecting the spectroscopic features such as the quantum efficiency of P(r) →P(fr) conversion, fluorescence, or the Resonance-Raman signature of the chromophore. Overall, the PAS-less photosensory module of Cph2 resembles Cph1 including its mode of isomerisation, but the P(fr) state is unstable.

Dwelling in the dark: procedures for the crystallography of phytochromes and other photochromic proteins.

Crystallization of phytochromes and other photochromic proteins is hampered by the conformational changes that they undergo on exposure to light. As a canonical phytochrome, cyanobacterial Cph1 switches between two stable states upon absorption of red/far-red light. Consequently, it is mandatory to work in darkness from protein purification to crystal cryoprotection in order to ensure complete occupancy of one state or the other. With the simple and inexpensive methods that have been developed, phytochromes and other photochromic molecules can effectively be handled and crystallized, as has been demonstrated by the solution of the three-dimensional structure of the Cph1 sensory module.

Chromophore structure of cyanobacterial phytochrome Cph1 in the Pr state: reconciling structural and spectroscopic data by QM/MM calculations.

A quantum mechanics (QM)/molecular mechanics (MM) hybrid method was applied to the Pr state of the cyanobacterial phytochrome Cph1 to calculate the Raman spectra of the bound PCB cofactor. Two QM/MM models were derived from the atomic coordinates of the crystal structure. The models differed in the protonation site of His(260) in the chromophore-binding pocket such that either the delta-nitrogen (M-HSD) or the epsilon-nitrogen (M-HSE) carried a hydrogen. The optimized structures of the two models display small differences specifically in the orientation of His(260) with respect to the PCB cofactor and the hydrogen bond network at the cofactor-binding site. For both models, the calculated Raman spectra of the cofactor reveal a good overall agreement with the experimental resonance Raman (RR) spectra obtained from Cph1 in the crystalline state and in solution, including Cph1 adducts with isotopically labeled PCB. However, a distinctly better reproduction of important details in the experimental spectra is provided by the M-HSD model, which therefore may represent an improved structure of the cofactor site. Thus, QM/MM calculations of chromoproteins may allow for refining crystal structure models in the chromophore-binding pocket guided by the comparison with experimental RR spectra. Analysis of the calculated and experimental spectra also allowed us to identify and assign the modes that sensitively respond to chromophore-protein interactions. The most pronounced effect was noted for the stretching mode of the methine bridge A-B adjacent to the covalent attachment site of PCB. Due a distinct narrowing of the A-B methine bridge bond angle, this mode undergoes a large frequency upshift as compared with the spectrum obtained by QM calculations for the chromophore in vacuo. This protein-induced distortion of the PCB geometry is the main origin of a previous erroneous interpretation of the RR spectra based on QM calculations of the isolated cofactor.

The structure of a complete phytochrome sensory module in the Pr ground state.

Phytochromes are red/far-red photochromic biliprotein photoreceptors, which in plants regulate seed germination, stem extension, flowering time, and many other light effects. However, the structure/functional basis of the phytochrome photoswitch is still unclear. Here, we report the ground state structure of the complete sensory module of Cph1 phytochrome from the cyanobacterium Synechocystis 6803. Although the phycocyanobilin (PCB) chromophore is attached to Cys-259 as expected, paralleling the situation in plant phytochromes but contrasting to that in bacteriophytochromes, the ZZZssa conformation does not correspond to that expected from Raman spectroscopy. We show that the PHY domain, previously considered unique to phytochromes, is structurally a member of the GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) family. Indeed, the tandem-GAF dumbbell revealed for phytochrome sensory modules is remarkably similar to the regulatory domains of cyclic nucleotide (cNMP) phosphodiesterases and adenylyl cyclases. A unique feature of the phytochrome structure is a long, tongue-like protrusion from the PHY domain that seals the chromophore pocket and stabilizes the photoactivated far-red-absorbing state (Pfr). The tongue carries a conserved PRxSF motif, from which an arginine finger points into the chromophore pocket close to ring D forming a salt bridge with a conserved aspartate residue. The structure that we present provides a framework for light-driven signal transmission in phytochromes.