Maria Andrea Mroginski

Mutational Analysis of Deinococcus radiodurans Bacteriophytochrome Reveals Key Amino Acids Necessary for the Photochromicity and Proton Exchange Cycle of Phytochromes

The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IXα (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-Rc-like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion.

Light-induced Proton Release of Phytochrome Is Coupled to the Transient Deprotonation of the Tetrapyrrole Chromophore

The Pr → Pfr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-RC were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in Pr and ZZEssa in Pfr, with all pyrrole nitrogens being protonated. Photoexcitation of Pr is followed by (at least) three thermal relaxation components in the formation of Pfr with time constants of 230 μs and 3.1 and 260 ms. H2O/D2O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-RC, respectively. Resonance Raman measurements of Meta-RC indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-RC are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the Pr → Pfr photocon-version mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the Pfr formation that switch between the active and the inactive state of the photoreceptor.

Highly conserved residues Asp-197 and His-250 in agp1 phytochrome control the proton affinity of the chromophore and Pfr formation

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pKa from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photo-induced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc → Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.

Molecular dynamics simulations of the adsorption of bone morphogenetic protein-2 on surfaces with medical relevance.

Bone morphogenetic protein-2 (BMP-2) plays a crucial role in osteoblast differentiation and proliferation. Its effective therapeutic use for ectopic bone and cartilage regeneration depends, among other factors, on the interaction with the carrier at the implant site. In this study, we used classical molecular dynamics (MD) and a hybrid approach of steered molecular dynamics (SMD) combined with MD simulations to investigate the initial stages of the adsorption of BMP-2 when approaching two implant surfaces, hydrophobic graphite and hydrophilic titanium dioxide rutile. Surface adsorption was evaluated for six different orientations of the protein, two end-on and four side-on, in explicit water environment. On graphite, we observed a weak but stable adsorption. Depending on the initial orientation, hydrophobic patches as well as flexible loops of the protein were involved in the interaction with graphite. On the contrary, BMP-2 adsorbed only loosely to hydrophilic titanium dioxide. Despite a favorable interaction energy between protein and the TiO(2) surface, the rapid formation of a two-layer water structure prevented the direct interaction between protein and titanium dioxide. The first water adlayer had a strong repulsive effect on the protein, while the second attracted the protein toward the surface. For both surfaces, hydrophobic graphite and hydrophilic titanium dioxide, denaturation of BMP-2 induced by adsorption was not observed on the nanosecond time scale.

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

Photointerconversion between the red light-absorbing (Pr) form and the far-red light-absorbing (Pfr) form is the central feature that allows members of the phytochrome (Phy) superfamily to act as reversible switches in light perception. Whereas the chromophore structure and surrounding binding pocket of Pr have been described, those for Pfr have remained enigmatic for various technical reasons. Here we describe a novel pair of Phys from two thermophilic cyanobacteria, Synechococcus sp. OS-A and OS-B′, that overcome several of these limitations. Like other cyanobacterial Phys, SyA-Cph1 and SyB-Cph1 covalently bind the bilin phycocyanobilin via their cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains and then assume the photointerconvertible Pr and Pfr states with absorption maxima at 630 and 704 nm, respectively. However, they are naturally missing the N-terminal Per/Arndt/Sim domain common to others in the Phy superfamily. Importantly, truncations containing only the GAF domain are monomeric, photochromic, and remarkably thermostable. Resonance Raman and NMR spectroscopy show that all four pyrrole ring nitrogens of phycocyanobilin are protonated both as Pr and following red light irradiation, indicating that the GAF domain by itself can complete the Pr to Pfr photocycle. 1H-15N two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters.

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 protonation state of Glu181 in rhodopsin revisited: interpretation of experimental data on the basis of QM/MM calculations.

The structure and spectroscopy of rhodopsin have been intensely studied in the past decade both experimentally and theoretically; however, important issues still remain unresolved. Of central interest is the protonation state of Glu181, where controversial and contradictory experimental evidence has appeared. While FTIR measurements indicate this residue to be unprotonated, preresonance Raman and UV-vis spectra have been interpreted in favor of a protonated Glu181. Previous computational approaches were not able to resolve this issue, providing contradicting data as well. Here, we perform hybrid QM/MM calculations using DFT methods for the electronic ground state, MRCI methods for the electronically excited states, and a polarization model for the MM part in order to investigate this issue systematically. We constructed various active-site models for protonated as well as unprotonated Glu181, which were evaluated by computing NMR, IR, Raman, and UV-vis spectroscopic data. The resulting differences in the UV-vis and Raman spectra between protonated and unprotonated models are very subtle, which has two major consequences. First, the common interpretation of prior Raman and UV-vis experiments in favor of a neutral Glu181 appears questionable, as it is based on the assumption that a charge at the Glu181 location would have a sizable impact. Second, also theoretical results should be interpreted with care. Spectroscopic differences between the structural models must be related to modeling uncertainties and intrinsic methodological errors. Despite a detailed comparison of various rhodopsins and mutants and consistently favorite results with charged Glu181 models, we find merely weak evidence from UV-vis and Raman calculations. On the contrary, difference FTIR and NMR chemical shift measurements on Rh mutants are indicative of the protonation state of Glu181. Supported by our results, they provide strong and independent evidence for a charged Glu181.

Structure of the Biliverdin Cofactor in the Pfr State of Bathy and Prototypical Phytochromes

Background: The Pr and Pfr parent states of prototypical and bathy bacteriophytochromes exhibit different thermal stabilities. Results: Unlike bathy phytochromes, the biliverdin cofactor of prototypical phytochromes displays distinct conformational heterogeneity in Pfr. Conclusion: This heterogeneity enables thermal Pfr to Pr conversion in prototypical phytochromes. Significance: Understanding thermal deactivation of the signaling Pfr state is essential for elucidating the molecular function of phytochromes. Phytochromes act as photoswitches between the red- and far-red absorbing parent states of phytochromes (Pr and Pfr). Plant phytochromes display an additional thermal conversion route from the physiologically active Pfr to Pr. The same reaction pattern is found in prototypical biliverdin-binding bacteriophytochromes in contrast to the reverse thermal transformation in bathy bacteriophytochromes. However, the molecular origin of the different thermal stabilities of the Pfr states in prototypical and bathy bacteriophytochromes is not known. We analyzed the structures of the chromophore binding pockets in the Pfr states of various bathy and prototypical biliverdin-binding phytochromes using a combined spectroscopic-theoretical approach. For the Pfr state of the bathy phytochrome from Pseudomonas aeruginosa, the very good agreement between calculated and experimental Raman spectra of the biliverdin cofactor is in line with important conclusions of previous crystallographic analyses, particularly the ZZEssa configuration of the chromophore and its mode of covalent attachment to the protein. The highly homogeneous chromophore conformation seems to be a unique property of the Pfr states of bathy phytochromes. This is in sharp contrast to the Pfr states of prototypical phytochromes that display conformational equilibria between two sub-states exhibiting small structural differences at the terminal methine bridges A-B and C-D. These differences may mainly root in the interactions of the cofactor with the highly conserved Asp-194 that occur via its carboxylate function in bathy phytochromes. The weaker interactions via the carbonyl function in prototypical phytochromes may lead to a higher structural flexibility of the chromophore pocket opening a reaction channel for the thermal (ZZE → ZZZ) Pfr to Pr back-conversion.

Photoconversion mechanism of the second GAF domain of cyanobacteriochrome AnPixJ and the cofactor structure of its green-absorbing state.

Cyanobacteriochromes are members of the phytochrome superfamily. In contrast to classical phytochromes, these small photosensors display a considerable variability of electronic absorption maxima. We have studied the light-induced conversions of the second GAF domain of AnPixJ, AnPixJg2, a phycocyanobilin-binding protein from the cyanobacterium Anabaena PCC 7120, using low-temperature resonance Raman spectroscopy combined with molecular dynamics simulations. AnPixJg2 is formed biosynthetically as a red-absorbing form (Pr) and can be photoconverted into a green-absorbing form (Pg). Forward and backward phototransformations involve the same reaction sequences and intermediates of similar cofactor structures as the corresponding processes in canonical phytochromes, including a transient cofactor deprotonation. Whereas the cofactor of the Pr state shows far-reaching similarities to the Pr states of classical phytochromes, the Pg form displays significant upshifts of the methine bridge stretching frequencies concomitant to the hypsochromically shifted absorption maximum. However, the cofactor in Pg is protonated and adopts a conformation very similar to the Pfr state of classical phytochromes. The spectral differences are probably related to an increased solvent accessibility of the chromophore which may reduce the π-electron delocalization in the phycocyanobilin and thus raise the energies of the first electronic transition and the methine bridge stretching modes. Molecular dynamics simulations suggest that the Z → E photoisomerization of the chromophore at the C-D methine bridge alters the interactions with the nearby Trp90 which in turn may act as a gate, allowing the influx of water molecules into the chromophore pocket. Such a mechanism of color tuning AnPixJg2 is unique among the cyanobacteriochromes studied so far.

Structure of the chromophore binding pocket in the Pr state of plant phytochrome phyA.

A homology structural model was generated for plant phytochrome phyA utilizing the crystal structure of the sensory module of cyanobacterial phytochrome Cph1 (Cph1Δ2). As chromophores, either the native phytochromobilin cofactor (PΦB) or phycocyanobilin (PCB), the natural cofactor in Cph1, was incorporated. These homology models were further optimized by molecular dynamics (MD) simulations revealing a satisfying overall agreement with the crystal structure of Cph1Δ2. Notable differences in the PΦB adduct of phyA result from a restructuring of the small helical segment α(7) that leads to displacements of a few amino acids away from the cofactor. This repositioning of residues also include aspartate 218 such that, instead of its carbonyl function as in Cph1Δ2, an additional water molecule forms hydrogen bonds with the ring B and C NH groups. To validate the phyA structural model in the chromophore binding pocket, Raman spectra of the cofactor were calculated by means of the quantum mechanics/molecular mechanics (QM/MM) hybrid methodology and compared with the experimental resonance Raman (RR) spectra. The satisfactory overall agreement between calculated and experimental spectra is taken as an indication for the good quality of the structural model. Moreover, the methine bridge stretching modes and the effects of isotopic labeling at selected positions of the chromophore are very well reproduced to allow confirming even details of the methine bridge geometry as predicted by the homology model. Specifically, it is demonstrated that the experimental RR spectra are consistent with a torsional angle of ring D with respect to ring C that is distinctly higher for phyA-PCB (45°) and phyA-PΦB (42°) than for Cph1Δ2 (30°). Raman spectra calculated from different points of the MD trajectory display variations of the mode frequencies and intensities reflecting the structural fluctuations from snapshot to snapshot. The snapshot spectrum of the lowest energy structure and the sum of all snapshot spectra afford an equally good description of the experimental data. Particularly large variations between the snapshots are noted for the N-H in-plane bending mode of the pyrrole rings B and C, which reflect alterations of the hydrogen bond interactions brought about by fluctuations of water molecules in the cofactor cavity. This overestimation of the water molecule mobility is a consequence of the deficiency of the current QM/MM methodology that, due to the lack of appropriate protein force fields, cannot adequately account for the electrostatics in the cofactor pocket.