Conserved histidine and tyrosine determine spectral responses through the water network in Deinococcus radiodurans phytochrome.

Phytochromes are red light-sensing photoreceptor proteins that bind a bilin chromophore. Here, we investigate the role of a conserved histidine (H260) and tyrosine (Y263) in the chromophore-binding domain (CBD) of Deinococcus radiodurans phytochrome (DrBphP). Using crystallography, we show that in the H260A variant, the missing imidazole side chain leads to increased water content in the binding pocket. On the other hand, Y263F mutation reduces the water occupancy around the chromophore. Together, these changes in water coordination alter the protonation and spectroscopic properties of the biliverdin. These results pinpoint the importance of this conserved histidine and tyrosine, and the related water network, for the function and applications of phytochromes.

Signal amplification and transduction in phytochrome photosensors.

Sensory proteins must relay structural signals from the sensory site over large distances to regulatory output domains. Phytochromes are a major family of red-light-sensing kinases that control diverse cellular functions in plants, bacteria and fungi. Bacterial phytochromes consist of a photosensory core and a carboxy-terminal regulatory domain. Structures of photosensory cores are reported in the resting state and conformational responses to light activation have been proposed in the vicinity of the chromophore. However, the structure of the signalling state and the mechanism of downstream signal relay through the photosensory core remain elusive. Here we report crystal and solution structures of the resting and activated states of the photosensory core of the bacteriophytochrome from Deinococcus radiodurans. The structures show an open and closed form of the dimeric protein for the activated and resting states, respectively. This nanometre-scale rearrangement is controlled by refolding of an evolutionarily conserved 'tongue', which is in contact with the chromophore. The findings reveal an unusual mechanism in which atomic-scale conformational changes around the chromophore are first amplified into an ångstrom-scale distance change in the tongue, and further grow into a nanometre-scale conformational signal. The structural mechanism is a blueprint for understanding how phytochromes connect to the cellular signalling network.

On the (un)coupling of the chromophore, tongue interactions, and overall conformation in a bacterial phytochrome.

Phytochromes are photoreceptors in plants, fungi, and various microorganisms and cycle between metastable red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Their light responses are thought to follow a conserved structural mechanism that is triggered by isomerization of the chromophore. Downstream structural changes involve refolding of the so-called tongue extension of the phytochrome-specific GAF-related (PHY) domain of the photoreceptor. The tongue is connected to the chromophore by conserved DIP and PRXSF motifs and a conserved tyrosine, but the role of these residues in signal transduction is not clear. Here, we examine the tongue interactions and their interplay with the chromophore by substituting the conserved tyrosine (Tyr263) in the phytochrome from the extremophile bacterium Deinococcus radiodurans with phenylalanine. Using optical and FTIR spectroscopy, X-ray solution scattering, and crystallography of chromophore-binding domain (CBD) and CBD-PHY fragments, we show that the absence of the Tyr263 hydroxyl destabilizes the ß-sheet conformation of the tongue. This allowed the phytochrome to adopt an α-helical tongue conformation regardless of the chromophore state, hence distorting the activity state of the protein. Our crystal structures further revealed that water interactions are missing in the Y263F mutant, correlating with a decrease of the photoconversion yield and underpinning the functional role of Tyr263 in phytochrome conformational changes. We propose a model in which isomerization of the chromophore, refolding of the tongue, and globular conformational changes are represented as weakly coupled equilibria. The results also suggest that the phytochromes have several redundant signaling routes.

Chromophore-Protein Interplay during the Phytochrome Photocycle Revealed by Step-Scan FTIR Spectroscopy.

Phytochrome proteins regulate many photoresponses of plants and microorganisms. Light absorption causes isomerization of the biliverdin chromophore, which triggers a series of structural changes to activate the signaling domains of the protein. However, the structural changes are elusive, and therefore the molecular mechanism of signal transduction remains poorly understood. Here, we apply two-color step-scan infrared spectroscopy to the bacteriophytochrome from Deinococcus radiodurans. We show by recordings in H2O and D2O that the hydrogen bonds to the biliverdin D-ring carbonyl become disordered in the first intermediate (Lumi-R) forming a dynamic microenvironment, then completely detach in the second intermediate (Meta-R), and finally reform in the signaling state (Pfr). The spectra reveal via isotope labeling that the refolding of the conserved "PHY-tongue" region occurs with the last transition between Meta-R and Pfr. Additional changes in the protein backbone are detected already within microseconds in Lumi-R. Aided by molecular dynamics simulations, we find that a strictly conserved salt bridge between an arginine of the PHY tongue and an aspartate of the chromophore binding domains is broken in Lumi-R and the arginine is recruited to the D-ring C═O. This rationalizes how isomerization of the chromophore is linked to the global structural rearrangement in the sensory receptor. Our findings advance the structural understanding of phytochrome photoactivation.

Origins of fluorescence in evolved bacteriophytochromes.

Use of fluorescent proteins to study in vivo processes in mammals requires near-infrared (NIR) biomarkers that exploit the ability of light in this range to penetrate tissue. Bacteriophytochromes (BphPs) are photoreceptors that couple absorbance of NIR light to photoisomerization, protein conformational changes, and signal transduction. BphPs have been engineered to form NIR fluorophores, including IFP1.4, Wi-Phy, and the iRFP series, initially by replacement of Asp-207 by His. This position was suggestive because its main chain carbonyl is within hydrogen-bonding distance to pyrrole ring nitrogens of the biliverdin chromophore, thus potentially functioning as a crucial transient proton sink during photoconversion. To explain the origin of fluorescence in these phytofluors, we solved the crystal structures of IFP1.4 and a comparison non-fluorescent monomeric phytochrome DrCBDmon. Met-186 and Val-288 in IFP1.4 are responsible for the formation of a tightly packed hydrophobic hub around the biliverdin D ring. Met-186 is also largely responsible for the blue-shifted IFP1.4 excitation maximum relative to the parent BphP. The structure of IFP1.4 revealed decreased structural heterogeneity and a contraction of two surface regions as direct consequences of side chain substitutions. Unexpectedly, IFP1.4 with Asp-207 reinstalled (IFPrev) has a higher fluorescence quantum yield (∼9%) than most NIR phytofluors published to date. In agreement, fluorescence lifetime measurements confirm the exceptionally long excited state lifetimes, up to 815 ps, in IFP1.4 and IFPrev. Our research helps delineate the origin of fluorescence in engineered BphPs and will facilitate the wide-spread adoption of phytofluors as biomarkers.

Connection between absorption properties and conformational changes in Deinococcus radiodurans phytochrome.

Phytochromes consist of several protein domains and a linear tetrapyrrole molecule, which interact as a red-light-sensing system. In this study, size-exclusion chromatography and light-scattering techniques are combined with UV-vis spectroscopy to investigate light-induced changes in dimeric Deinococcus radiodurans bacterial phytochrome (DrBphP) and its subdomains. The photosensory unit (DrCBD-PHY) shows an unusually stable Pfr state with minimal dark reversion, whereas the histidine kinase (HK) domain facilitates dark reversion to the resting state. Size-exclusion chromatography reveals that all phytochrome fragments remain as dimers in the illuminated state and dark state. Still, the elution profiles of all phytochrome fragments differ between the illuminated and dark states. The differences are observed reliably only when the whole UV-vis spectrum is characterized along the elution profile and show more Pfr-state characteristics at later elution volumes in DrBphP and DrCBD-PHY fragments. This implies that the PHY domain has an important role in amplifying and relaying light-induced conformational changes to the HK domain. In the illuminated state, the HK domain appears partially unfolded and prone to form oligomers. The oligomerization of DrBphP can be diminished by converting the molecule back to the resting Pr state by using far-red light.

Excitation energy transfer in the LHC-II trimer: from carotenoids to chlorophylls in space and time.

Exciton model for description of experimentally determined excitation energy transfer from carotenoids to chlorophylls in the LHC-II trimer of spinach is presented. Such an approach allows connecting the excitonic states to the spatial structure of the complex and hence descriptions of advancements of the initially created excitations in space and time. Carotenoids were excited at 490 nm and at 500 nm and induced absorbance changes probed in the Chl Q(y) region to provide kinetic data that were interpreted by using the results from exciton calculations. Calculations included the 42 chlorophylls and the 12 carotenoids of the complex, Soret, Q(x) and Q(y) states of the chlorophylls, and the main absorbing S(2) state of the carotenoids. According to the calculations excitation at 500 nm populates mostly a mixed Lut S(2) Chl a Soret state, from where excitation is transferred to the Q(x) and Q(y) states of the Chl a's on the stromal side. Internal conversion of the mixed state to a mixed Lut S(1) and Chl a Q(y) state provides a channel for Lut S(1) to Chl a Q(y) energy transfer. The results from the calculations support a picture where excitation at 490 nm populates primarily a mixed neoxanthin S(2) Chl b Soret state. From this state excitation from neoxanthin is transferred to iso-energetic Chl b Soret states or via internal conversion to S(1) Chl b Q(y) states. From the Soret states excitation proceeds via internal conversion to Q(y) states of Chl b's mostly on the lumenal side. A rapid Chl b to Chl a transfer and subsequent transfer to the stromal side Chl a's and to the final state completes the process after 490 nm excitation. The interpretation is further supported by the fact that excitation energy transfer kinetics after excitation of neoxanthin at 490 nm and the Chl b Q(y) band at 647 nm (Linnanto et al., Photosynth Res 87:267-279, 2006) are very similar.

Retene, pyrene and phenanthrene cause distinct molecular-level changes in the cardiac tissue of rainbow trout (Oncorhynchus mykiss) larvae, part 2 - Proteomics and metabolomics.

Polycyclic aromatic hydrocarbons (PAHs) are global contaminants of concern. Despite several decades of research, their mechanisms of toxicity are not very well understood. Early life stages of fish are particularly sensitive with the developing cardiac tissue being a main target of PAHs toxicity. The mechanisms of cardiotoxicity of the three widespread model polycyclic aromatic hydrocarbons (PAHs) retene, pyrene and phenanthrene were explored in rainbow trout (Oncorhynchus mykiss) early life stages. Newly hatched larvae were exposed to sublethal doses of each individual PAH causing no detectable morphometric alterations. Changes in the cardiac proteome and metabolome were assessed after 7 or 14 days of exposure to each PAH. Phase I and II enzymes regulated by the aryl hydrocarbon receptor were significantly induced by all PAHs, with retene being the most potent compound. Retene significantly altered the level of several proteins involved in key cardiac functions such as muscle contraction, cellular tight junctions or calcium homeostasis. Those findings were quite consistent with previous reports regarding the effects of retene on the cardiac transcriptome. Significant changes in proteins linked to iron and heme metabolism were observed following exposure to pyrene. While phenanthrene also altered the levels of several proteins in the cardiac tissue, no clear mechanisms or pathways could be highlighted. Due to high variability between samples, very few significant changes were detected in the cardiac metabolome overall. Slight but significant changes were still observed for pyrene and phenanthrene, suggesting possible effects on several energetic or signaling pathways. This study shows that early exposure to different PAHs can alter the expression of key proteins involved in the cardiac function, which could potentially affect negatively the fitness of the larvae and later of the juvenile fish.

Retene, pyrene and phenanthrene cause distinct molecular-level changes in the cardiac tissue of rainbow trout (Oncorhynchus mykiss) larvae, part 1 - Transcriptomics.

Polycyclic aromatic hydrocarbons (PAHs) are contaminants of concern that impact every sphere of the environment. Despite several decades of research, their mechanisms of toxicity are still poorly understood. This study explores the mechanisms of cardiotoxicity of the three widespread model PAHs retene, pyrene and phenanthrene in the rainbow trout (Oncorhynchus mykiss) early life stages. Newly hatched larvae were exposed to each individual compound at sublethal doses causing no significant increase in the prevalence of deformities. Changes in the cardiac transcriptome were assessed after 1, 3, 7 and 14 days of exposure using custom Salmo salar microarrays. The highest number of differentially expressed genes was observed after 1 or 3 days of exposure, and retene was the most potent compound in that regard. Over-representation analyses suggested that genes related to cardiac ion channels, calcium homeostasis and muscle contraction (actin binding, troponin and myosin complexes) were especially targeted by retene. Pyrene was also able to alter similar myosin-related genes, but at a different timing and in an opposite direction, suggesting compound-specific mechanisms of toxicity. Pyrene and to a lesser extent phenanthrene were altering key genes linked to the respiratory electron transport chain and to oxygen and iron metabolism. Overall, phenanthrene was not very potent in inducing changes in the cardiac transcriptome despite being apparently metabolized at a slower rate than retene and pyrene. The present study shows that exposure to different PAHs during the first few days of the swim-up stage can alter the expression of key genes involved into the cardiac development and function, which could potentially affect negatively the fitness of the larvae in the long term.

Exciplex mediated photoinduced electron transfer reactions of phthalocyanine-fullerene dyads.

Evidences of an intramolecular exciplex intermediate in a photoinduced electron transfer (ET) reaction of double-linked free-base and zinc phthalocyanine-C60 dyads were found. This was the first time for a dyad with phthalocyanine donor. Excitation of the phthalocyanine moiety of the dyads results in rapid ET from phthalocyanine to fullerene via an exciplex state in both polar and nonpolar solvents. Relaxation of the charge-separated (CS) state Pc(*+)-C60(*-) in a polar solvent occurs directly to the ground state in 30-70 ps. In a nonpolar solvent, roughly 20% of the molecules undergo transition from the CS state to phthalocyanine triplet state (3)Pc*-C60 before relaxation to the ground state. Formation of the CS state was confirmed with electron spin resonance measurements at low temperature in both polar and nonpolar solvent. Reaction schemes for the photoinduced ET reactions of the dyads were completed with rate constants obtained from the time-resolved absorption and emission measurements and with state energies obtained from the fluorescence, phosphorescence, and voltammetric measurements.

Size- and Wavelength-Dependent Two-Photon Absorption Cross-Section of CsPbBr3 Perovskite Quantum Dots.

All-inorganic colloidal perovskite quantum dots (QDs) based on cesium, lead, and halide have recently emerged as promising light emitting materials. CsPbBr3 QDs have also been demonstrated as stable two-photon-pumped lasing medium. However, the reported two photon absorption (TPA) cross sections for these QDs differ by an order of magnitude. Here we present an in-depth study of the TPA properties of CsPbBr3 QDs with mean size ranging from 4.6 to 11.4 nm. By using femtosecond transient absorption (TA) spectroscopy we found that TPA cross section is proportional to the linear one photon absorption. The TPA cross section follows a power law dependence on QDs size with exponent 3.3 ± 0.2. The empirically obtained power-law dependence suggests that the TPA process through a virtual state populates exciton band states. The revealed power-law dependence and the understanding of TPA process are important for developing high performance nonlinear optical devices based on CsPbBr3 nanocrystals.

Light-induced structural changes in a monomeric bacteriophytochrome.

Phytochromes sense red light in plants and various microorganism. Light absorption causes structural changes within the protein, which alter its biochemical activity. Bacterial phytochromes are dimeric proteins, but the functional relevance of this arrangement remains unclear. Here, we use time-resolved X-ray scattering to reveal the solution structural change of a monomeric variant of the photosensory core module of the phytochrome from Deinococcus radiodurans. The data reveal two motions, a bend and a twist of the PHY domain with respect to the chromophore-binding domains. Infrared spectroscopy shows the refolding of the PHY tongue. We conclude that a monomer of the phytochrome photosensory core is sufficient to perform the light-induced structural changes. This implies that allosteric cooperation with the other monomer is not needed for structural activation. The dimeric arrangement may instead be intrinsic to the biochemical output domains of bacterial phytochromes.

Structural photoactivation of a full-length bacterial phytochrome.

Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro- and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes.

The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography.

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.

Fast Photochemistry of Prototypical Phytochromes-A Species vs. Subunit Specific Comparison.

Phytochromes are multi-domain red light photosensor proteins, which convert red light photons to biological activity utilizing the multitude of structural and chemical reactions. The steady increase in structural information obtained from various bacteriophytochromes has increased understanding about the functional mechanism of the photochemical processes of the phytochromes. Furthermore, a number of spectroscopic studies have revealed kinetic information about the light-induced reactions. The spectroscopic changes are, however, challenging to connect with the structural changes of the chromophore and the protein environment, as the excited state properties of the chromophores are very sensitive to the small structural and chemical changes of their environment. In this article, we concentrate on the results of ultra-fast spectroscopic experiments which reveal information about the important initial steps of the photoreactions of the phytochromes. We survey the excited state properties obtained during the last few decades. The differences in kinetics between different research laboratories are traditionally related to the differences of the studied species. However, we notice that the variation in the excited state properties depends on the subunit composition of the protein as well. This observation illustrates a feedback mechanism from the other domains to the chromophore. We propose that two feedback routes exist in phytochromes between the chromophore and the remotely located effector domain. The well-known connection between the subunits is the so-called tongue region, which changes its secondary structure while changing the light-activated state of the system. The other feedback route which we suggest is less obvious, it is made up of several water molecules ranging from the dimer interface to the vicinity of the chromophore, allowing even proton transfer reactions nearby the chromophore.

Removal of Chromophore-Proximal Polar Atoms Decreases Water Content and Increases Fluorescence in a Near Infrared Phytofluor.

Genetically encoded fluorescent markers have revolutionized cell and molecular biology due to their biological compatibility, controllable spatiotemporal expression, and photostability. To achieve in vivo imaging in whole animals, longer excitation wavelength probes are needed due to the superior ability of near infrared light to penetrate tissues unimpeded by absorbance from biomolecules or autofluorescence of water. Derived from near infrared-absorbing bacteriophytochromes, phytofluors are engineered to fluoresce in this region of the electromagnetic spectrum, although high quantum yield remains an elusive goal. An invariant aspartate residue is of utmost importance for photoconversion in native phytochromes, presumably due to the proximity of its backbone carbonyl to the pyrrole ring nitrogens of the biliverdin (BV) chromophore as well as the size and charge of the side chain. We hypothesized that the polar interaction network formed by the charged side chain may contribute to the decay of the excited state via proton transfer. Thus, we chose to further probe the role of this amino acid by removing all possibility for polar interactions with its carboxylate side chain by incorporating leucine instead. The resultant fluorescent protein, WiPhy2, maintains BV binding, monomeric status, and long maximum excitation wavelength while minimizing undesirable protoporphyrin IXα binding in cells. A crystal structure and time-resolved fluorescence spectroscopy reveal that water near the BV chromophore is excluded and thus validate our hypothesis that removal of polar interactions leads to enhanced fluorescence by increasing the lifetime of the excited state. This new phytofluor maintains its fluorescent properties over a broad pH range and does not suffer from photobleaching. WiPhy2 achieves the best compromise to date between high fluorescence quantum yield and long illumination wavelength in this class of fluorescent proteins.

Ubiquitous Structural Signaling in Bacterial Phytochromes.

The phytochrome family of light-switchable proteins has long been studied by biochemical, spectroscopic and crystallographic means, while a direct probe for global conformational signal propagation has been lacking. Using solution X-ray scattering, we find that the photosensory cores of several bacterial phytochromes undergo similar large-scale structural changes upon red-light excitation. The data establish that phytochromes with ordinary and inverted photocycles share a structural signaling mechanism and that a particular conserved histidine, previously proposed to be involved in signal propagation, in fact tunes photoresponse.

The structural diversity of benzofuran resorcinarene leads to enhanced fluorescence.

An unexpected and previously unknown resorcinarene mono-crown with a fused benzofuran moiety in its macrocyclic core was obtained as a byproduct from a bridging reaction of tetramethoxy resorcinarene with tetraethylene glycol ditosylate. The formation of the fused benzofuran moiety in the resorcinarene macrocycle resulted in a unique rigid and puckered boat conformation, as shown by XRD studies in the solid state. Modification of the macrocycle was also observed to affect the photophysical properties in solution by enhancing the fluorescence brightness compared with a conventional resorcinarene macrocycle. The fluorescent properties enabled unique detection of structural features, that is, the rigid boat conformation with the conjugated benzofuran system and the more flexible crown bridge part, in solution.

Subcellular localization of bacteriophage PRD1 proteins in Escherichia coli.

Bacteria possess an intricate internal organization resembling that of the eukaryotes. The complexity is especially prominent at the bacterial cell poles, which are also known to be the preferable sites for some bacteriophages to infect. Bacteriophage PRD1 is a well-known model serving as an ideal system to study structures and functions of icosahedral internal membrane-containing viruses. Our aim was to analyze the localization and interactions of individual PRD1 proteins in its native host Escherichia coli. This was accomplished by constructing a vector library for production of fluorescent fusion proteins. Analysis of solubility and multimericity of the fusion proteins, as well as their localization in living cells by confocal microscopy, indicated that multimeric PRD1 proteins were prone to localize in the cell poles. Furthermore, PRD1 spike complex proteins P5 and P31, as fusion proteins, were shown to be functional in the virion assembly. In addition, they were shown to co-localize in the specific polar area of the cells, which might have a role in the multimerization and formation of viral protein complexes.