Merocyanines form bacteriorhodopsins with strongly bathochromic absorption maxima.

There is a need to shift the absorbance of biomolecules to the optical transparency window of tissue for applications in optogenetics and photo-pharmacology. There are a few strategies to achieve the so-called red shift of the absorption maxima. Herein, a series of 11 merocyanine dyes were synthesized and employed as chromophores in place of retinal in bacteriorhodopsin (bR) to achieve a bathochromic shift of the absorption maxima relative to bR's [Formula: see text] of 568 nm. Assembly with the apoprotein bacterioopsin (bO) led to stable, covalently bound chromoproteins with strongly bathochromic absorbance bands, except for three compounds. Maximal red shifts were observed for molecules 9, 2, and 8 in bR where the [Formula: see text] was 766, 755, and 736 nm, respectively. While these three merocyanines have different end groups, they share a similar structural feature, namely, a methyl group which is located at the retinal equivalent position 13 of the polyene chain. The absorption and fluorescence data are also presented for the retinal derivatives in their aldehyde, Schiff base (SB), and protonated SB (PSB) forms in solution. According to their hemicyanine character, the PSBs and their analogue bRs exhibited fluorescence quantum yields (Φf) several orders of magnitude greater than native bR (Φf 0.02 to 0.18 versus 1.5 × 10-5 in bR) while also exhibiting much smaller Stokes shifts than bR (400 to 1000 cm-1 versus 4030 cm-1 in bR). The experimental results are complemented by quantum chemical calculations where excellent agreement between the experimental [Formula: see text] and the calculated [Formula: see text] was achieved with the second-order algebraic-diagrammatic construction [ADC(2)] method. In addition, quantum mechanics/molecular mechanics (QM/MM) calculations were employed to shed light on the origin of the bathochromic shift of merocyanine 2 in bR compared with native bR.

Conserved tyrosine in phytochromes controls the photodynamics through steric demand and hydrogen bonding capabilities.

Using ultrafast spectroscopy and site-specific mutagenesis, we demonstrate the central role of a conserved tyrosine within the chromophore binding pocket in the forward (Pr â†’ Pfr) photoconversion of phytochromes. Taking GAF1 of the knotless phytochrome All2699g1 from Nostoc as representative member of phytochromes, it was found that the mutations have no influence on the early (<30 ps) dynamics associated with conformational changes of the chromophore in the excited state. Conversely, they drastically impact the extended protein-controlled excited state decay (>100 ps). Thus, the steric demand, position and H-bonding capabilities of the identified tyrosine control the chromophore photoisomerization while leaving the excited state chromophore dynamics unaffected. In effect, this residue operates as an isomerization-steric-gate that tunes the excited state lifetime and the photoreaction efficiency by modulating the available space of the chromophore and by stabilizing the primary intermediate Lumi-R. Understanding the role of such a conserved structural element sheds light on a key aspect of phytochrome functionality and provides a basis for rational design of optimized photoreceptors for biotechnological applications.

Photocycle of a cyanobacteriochrome: a charge defect on ring C impairs conjugation in chromophore.

A large number of novel phytochromes named cyanobacteriochromes (CBCRs) have been recently identified. CBCRs appear to be attractive for further in-depth studies as paradigms for phytochromes because of their related photochemistry, but simpler domain architecture. Elucidating the mechanisms of spectral tuning for the bilin chromophore down to the molecular/atomic level is a prerequisite to design fine-tuned photoswitches for optogenetics. Several explanations for the blue shift during photoproduct formation associated with the red/green CBCRs represented by Slr1393g3 have been developed. There are, however, only sparse mechanistic data concerning the factors controlling stepwise absorbance changes along the reaction pathways from the dark state to the photoproduct and vice versa in this subfamily. Conventional cryotrapping of photocycle intermediates of phytochromes has proven experimentally intractable for solid-state NMR spectroscopy within the probe. Here, we have developed a simple method to circumvent this hindrance by incorporating proteins into trehalose glasses which allows four photocycle intermediates of Slr1393g3 to be isolated for NMR use. In addition to identifying the chemical shifts and chemical shift anisotropy principal values of selective chromophore carbons in various photocycle states, we generated QM/MM models of the dark state and photoproduct as well as of the primary intermediate of the backward-reaction. We find the motion of all three methine bridges in both reaction directions but in different orders. These molecular events channel light excitation to drive distinguishable transformation processes. Our work also suggests that polaronic self-trapping of a conjugation defect by displacement of the counterion during the photocycle would play a role in tuning the spectral properties of both the dark state and photoproduct.

Functional characterization of xanthorhodopsin in Salinivibrio socompensis, a novel halophile isolated from modern stromatolites.

A putative xanthorhodopsin-encoding gene, XR34, was found in the genome of the moderately halophilic gammaproteobacterium Salinivibrio socompensis S34, isolated from modern stromatolites found on the shore of Laguna Socompa (3570 m), Argentina Puna. XR-encoding genes were clustered together with genes encoding X-carotene, retinal (vitamin-A aldehyde), and carotenoid biosynthesis enzymes while the carotene ketolase gene critical for the salinixanthin antenna compound was absent. To identify its functional behavior, we herein overexpressed and characterized this intriguing microbial rhodopsin. Recombinant XR34 showed all the salient features of canonical microbial rhodopsin and covalently bound retinal as a functional chromophore with λmax = 561 nm (εmax ca. 60,000 M-1 cm-1). Two canonical counterions with pK values of around 6 and 3 were identified by pH titration of the recombinant protein. With a recovery time of approximately half an hour in the dark, XR34 shows light-dark adaptation shifting the absorption maximum from 551 to 561 nm. Laser-flash induced photochemistry at pH 9 (deprotonated primary counterion) identified a photocycle starting with a K-like intermediate, followed by an M-state (λmax ca. 400 nm, deprotonated Schiff base), and a final long wavelength-absorbing N- or O-like intermediate before returning to the parental 561 nm-state. Initiating the photocycle at pH 5 (protonated counterion) yields only bathochromic intermediates, due to the lacking capacity of the counterion to accept the Schiff base proton. Illumination of the membrane-embedded protein yielded a capacitive transport current. The presence of the M-intermediate under these conditions was demonstrated by a blue light-induced shunt process.

A red-green photochromic bacterial protein as a new contrast agent for improved photoacoustic imaging.

The GAF3 domain of the cyanobacteriochrome Slr1393 from Synechocystis sp. PCC6803, binding phycocyanobilin as a chromophore, shows photochromicity between two stable, green- and red-absorbing states, characterized by relatively high photoconversion yields. Using nanosecond-pulsed excitation by red or green light, respectively, and suitable cw photoconversion beams, we demonstrate that the light-modulatable photoacoustic waveforms arising from GAF3 can be easily distinguished from background signals originating from non-modulatable competitive absorbers and scattering media. It is demonstrated that this effect can be exploited to identify the position of the photochromic molecule by using as a phantom a cylindrical capillary tube filled with either a GAF3 solution or with an E.coli suspension overexpressing GAF3. These properties identify the high potential of GAF3 to be included in the palette of genetically encoded photochromic probes for photoacoustic imaging.

Light- and pH-dependent structural changes in cyanobacteriochrome AnPixJg2.

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensory proteins that play an essential role in regulating phototaxis, chromatic acclimation, and cell aggregation in cyanobacteria. Here, we apply solid-state NMR spectroscopy to the red/green GAF2 domain of the CBCR AnPixJ assembled in vitro with a uniformly 13C- and 15N-labeled bilin chromophore, tracking changes in electronic structure, geometry, and structural heterogeneity of the chromophore as well as intimate contacts between the chromophore and protein residues in the photocycle. Our data confirm that the bilin ring D is strongly twisted with respect to the B-C plane in both dark and photoproduct states. We also identify a greater structural heterogeneity of the bilin chromophore in the photoproduct than in the dark state. In addition, the binding pocket is more hydrated in the photoproduct. Observation of interfacial 1H contacts of the photoproduct chromophore, together with quantum mechanics/molecular mechanics (QM/MM)-based structural models for this photoproduct, clearly suggests the presence of a biprotonated (cationic) imidazolium side-chain for a conserved histidine residue (322) at a distance of ~2.7 Å, generalizing the recent theoretical findings that explicitly link the structural heterogeneity of the dark-state chromophore to the protonation of this specific residue. Moreover, we examine pH effects on this in vitro assembled holoprotein, showing a substantially altered electronic structure and protonation of the photoproduct chromophore even with a small pH drop from 7.8 to 7.2. Our studies provide further information regarding the light- and pH-induced changes of the chromophore and the rearrangements of the hydrogen-bonding and electrostatic interaction network around it. Possible correlations between structural heterogeneity of the chromophore, protonation of the histidine residue nearby, and hydration of the pocket in both photostates are discussed.

Proteomic Signatures of Microbial Adaptation to the Highest Ultraviolet-Irradiation on Earth: Lessons From a Soil Actinobacterium.

In the Central Andean region in South America, high-altitude ecosystems (3500-6000 masl) are distributed across Argentina, Chile, Bolivia, and Peru, in which poly-extremophilic microbes thrive under extreme environmental conditions. In particular, in the Puna region, total solar irradiation and UV incidence are the highest on Earth, thus, restraining the physiology of individual microorganisms and the composition of microbial communities. UV-resistance of microbial strains thriving in High-Altitude Andean Lakes was demonstrated and their mechanisms were partially characterized by genomic analysis, biochemical and physiological assays. Then, the existence of a network of physiological and molecular mechanisms triggered by ultraviolet light exposure was hypothesized and called "UV-resistome". It includes some or all of the following subsystems: (i) UV sensing and effective response regulators, (ii) UV-avoidance and shielding strategies, (iii) damage tolerance and oxidative stress response, (iv) energy management and metabolic resetting, and (v) DNA damage repair. Genes involved in the described UV-resistome were recently described in the genome of Nesterenkonia sp. Act20, an actinobacterium which showed survival to high UV-B doses as well as efficient photorepairing capability. The aim of this work was to use a proteomic approach together with photoproduct measurements to help dissecting the molecular events involved in the adaptive response of a model High-Altitude Andean Lakes (HAAL) extremophilic actinobacterium, Nesterenkonia sp. Act20, under artificial UV-B radiation. Our results demonstrate that UV-B exposure induced over-abundance of a well-defined set of proteins while recovery treatments restored the proteomic profiles present before the UV-challenge. The proteins involved in this complex molecular network were categorized within the UV-resistome subsystems: damage tolerance and oxidative stress response, energy management and metabolic resetting, and DNA damage repair.

The Red Edge: Bilin-Binding Photoreceptors as Optogenetic Tools and Fluorescence Reporters.

This review adds the bilin-binding phytochromes to the Chemical Reviews thematic issue "Optogenetics and Photopharmacology". The work is structured into two parts. We first outline the photochemistry of the covalently bound tetrapyrrole chromophore and summarize relevant spectroscopic, kinetic, biochemical, and physiological properties of the different families of phytochromes. Based on this knowledge, we then describe the engineering of phytochromes to further improve these chromoproteins as photoswitches and review their employment in an ever-growing number of different optogenetic applications. Most applications rely on the light-controlled complex formation between the plant photoreceptor PhyB and phytochrome-interacting factors (PIFs) or C-terminal light-regulated domains with enzymatic functions present in many bacterial and algal phytochromes. Phytochrome-based optogenetic tools are currently implemented in bacteria, yeast, plants, and animals to achieve light control of a wide range of biological activities. These cover the regulation of gene expression, protein transport into cell organelles, and the recruitment of phytochrome- or PIF-tagged proteins to membranes and other cellular compartments. This compilation illustrates the intrinsic advantages of phytochromes compared to other photoreceptor classes, e.g., their bidirectional dual-wavelength control enabling instant ON and OFF regulation. In particular, the long wavelength range of absorption and fluorescence within the "transparent window" makes phytochromes attractive for complex applications requiring deep tissue penetration or dual-wavelength control in combination with blue and UV light-sensing photoreceptors. In addition to the wide variability of applications employing natural and engineered phytochromes, we also discuss recent progress in the development of bilin-based fluorescent proteins.

Mapping the role of aromatic amino acids within a blue-light sensing LOV domain.

Photosensing LOV (Light, Oxygen, Voltage) domains detect and respond to UVA/Blue (BL) light by forming a covalent adduct between the flavin chromophore and a nearby cysteine, via the decay of the flavin triplet excited state. LOV domains where the reactive cysteine has been mutated are valuable fluorescent tools for microscopy and as genetically encoded photosensitisers for reactive oxygen species. Besides being convenient tools for applications, LOV domains without the reactive cysteine (naturally occurring or engineered) can still be functionally photoactivated via formation of a neutral flavin radical. Tryptophans and tyrosines are held as the main partners as potential electron donors to the flavin excited states. In this work, we explore the relevance of aromatic amino acids in determining the photophysical features of the LOV protein Mr4511 from Methylobacterium radiotolerans by introducing point mutations into the C71S variant that does not form the covalent adduct. By using an array of spectroscopic techniques we measured the fluorescence quantum yields and lifetimes, the triplet yields and lifetimes, and the efficiency of singlet oxygen (SO) formation for eleven Mr4511 variants. Insertion of Trp residues at distances between 0.6 and 1.5 nm from the flavin chromophore results in strong quenching of the flavin excited triplet state and, at the shorter distances even of the singlet excited state. The mutation F130W (ca. 0.6 nm) completely quenches the singlet excited state, preventing triplet formation: in this case, even if the cysteine is present, the photo-adduct is not formed. Tyrosines are also quenchers for the flavin excited states, although not as efficient as Trp residues, as demonstrated with their substitution with the inert phenylalanine. For one of these variants, C71S/Y116F, we found that the quantum yield of formation for singlet oxygen is 0.44 in aqueous aerobic solution, vs 0.17 for C71S. Based on our study with Mr4511 and on literature data for other LOV domains we suggest that Trp and Tyr residues too close to the flavin chromophore (at distances less than 0.9 nm) reduce the yield of photoproduct formation and that introduction of inert Phe residues in key positions can help in developing efficient, LOV-based photosensitisers.

A light life together: photosensing in the plant microbiota.

Bacteria and fungi of the plant microbiota can be phytopathogens, parasites or symbionts that establish mutually advantageous relationships with plants. They are often rich in photoreceptors for UVA-Visible light, and in many cases, they exhibit light regulation of growth patterns, infectivity or virulence, reproductive traits, and production of pigments and of metabolites. In addition to the light-driven effects, often demonstrated via the generation of photoreceptor gene knock-outs, microbial photoreceptors can exert effects also in the dark. Interestingly, some fungi switch their attitude towards plants in dependence of illumination or dark conditions in as much as they may be symbiotic or pathogenic. This review summarizes the current knowledge about the roles of light and photoreceptors in plant-associated bacteria and fungi aiming at the identification of common traits and general working ideas. Still, reports on light-driven infection of plants are often restricted to the description of macroscopically observable phenomena, whereas detailed information on the molecular level, e.g., protein-protein interaction during signal transduction or induction mechanisms of infectivity/virulence initiation remains sparse. As it becomes apparent from still only few molecular studies, photoreceptors, often from the red- and the blue light sensitive groups interact and mutually modulate their individual effects. The topic is of great relevance, even in economic terms, referring to plant-pathogen or plant-symbionts interactions, considering the increasing usage of artificial illumination in greenhouses, the possible light-regulation of the synthesis of plant-growth stimulating substances or herbicides by certain symbionts, and the biocontrol of pests by selected fungi and bacteria in a sustainable agriculture.

Effect of the PHY Domain on the Photoisomerization Step of the Forward Pr →Pfr Conversion of a Knotless Phytochrome.

Phytochrome photoreceptors operate via photoisomerization of a bound bilin chromophore. Their typical architecture consists of GAF, PAS and PHY domains. Knotless phytochromes lack the PAS domain, while retaining photoconversion abilities, with some being able to photoconvert with just the GAF domain. Therefore, we investigated the ultrafast photoisomerization of the Pr state of a knotless phytochrome to reveal the effect of the PHY domain and its "tongue" region on the transduction of the light signal. We show that the PHY domain does not affect the initial conformational dynamics of the chromophore. However, it significantly accelerates the consecutively induced reorganizational dynamics of the protein, necessary for the progression of the photoisomerization. Consequently, the PHY domain keeps the bilin and its binding pocket in a more reactive conformation, which decreases the extent of protein reorganization required for the chromophore isomerization. Thereby, less energy is lost along nonproductive reaction pathways, resulting in increased efficiency.

Lyophilization Reveals a Multitude of Structural Conformations in the Chromophore of a Cph2-like Phytochrome.

Cyanobacteria sense and respond to various colors of light employing a large number of bilin-based phytochrome-like photoreceptors. All2699 from Nostoc 7120 has three consecutive GAF domains with GAF1 and GAF3 binding a phycocyanobilin chromophore. GAF1, even when expressed independently, can be photoconverted between red-absorbing Pr and far-red-absorbing Pfr states, while the nonphotosensory GAF2 domain is structurally and functionally homologous to the PHY domains in canonical and Cph2-like phytochromes. Here, we characterize possible bilin chromophore conformers using solid-state NMR spectroscopy on the two lyophilized All2699 samples (GAF1-only and GAF1-PHY constructs). On the basis of complete 1H, 13C, and 15N assignments for the chromophore obtained on the two Pr lyophilizates, multiple static conformations of the chromophore in both cases are identified. Moreover, most atoms of the chromophore in the bidomain sample show only subtle changes in the mean chemical shifts relative to those in frozen solution (FS), indicating an optimized interaction of the GAF2 domain with the GAF1-bound chromophore. Our results confirm the conservation of key chromophore-protein interactions and the photoreversibility in both All2699 lyophilizates, offering the possibility to investigate conformational distributions of the heterogeneous chromophore and its functional consequences in phytochromes and other bilin-dependent photoreceptors intractable by the solid-state NMR technique as FSs.

Unequal twins: Unraveling the reaction mechanism of dimeric histidine kinases.

Histidine kinases (HKs), together with their partner proteins, the response regulators (RRs), form the ubiquitous two-component systems that are global players in control and adjustment of microbial lifestyle. Although their basic function (i.e. the transfer of a phosphate group from the HK to its RR partner) is simple to articulate, deciphering the molecular details of this process has proven anything but simple, especially when quantitative aspects come into play. Bouillet et al. report a series of elegant and sophisticated experiments to quantitatively understand HK functions, clearing up several open questions and providing a new strategy for future work in the field.

The interplay between chromophore and protein determines the extended excited state dynamics in a single-domain phytochrome.

Phytochromes are a diverse family of bilin-binding photoreceptors that regulate a wide range of physiological processes. Their photochemical properties make them attractive for applications in optogenetics and superresolution microscopy. Phytochromes undergo reversible photoconversion triggered by the Z ⇄ E photoisomerization about the double bond in the bilin chromophore. However, it is not fully understood at the molecular level how the protein framework facilitates the complex photoisomerization dynamics. We have studied a single-domain bilin-binding photoreceptor All2699g1 (Nostoc sp. PCC 7120) that exhibits photoconversion between the red light-absorbing (Pr) and far red-absorbing (Pfr) states just like canonical phytochromes. We present the crystal structure and examine the photoisomerization mechanism of the Pr form as well as the formation of the primary photoproduct Lumi-R using time-resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations. We show that the unusually long excited state lifetime (broad lifetime distribution centered at ∼300 picoseconds) is due to the interactions between the isomerizing pyrrole ring D and an adjacent conserved Tyr142. The decay kinetics shows a strongly distributed character which is imposed by the nonexponential protein dynamics. Our findings offer a mechanistic insight into how the quantum efficiency of the bilin photoisomerization is tuned by the protein environment, thereby providing a structural framework for engineering bilin-based optical agents for imaging and optogenetics applications.

Tongue Refolding in the Knotless Cyanobacterial Phytochrome All2699.

Phytochromes regulate central responses of plants and microorganisms such as shade avoidance and photosystem synthesis. Canonical phytochromes comprise a photosensory module of three domains. The C-terminal phytochrome-specific (PHY) domain interacts via a tongue element with the bilin chromophore in the central GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain. The bilin isomerizes upon illumination with red light, transforming the receptor from the Pr state to the Pfr state. The "knotless" phytochrome All2699 from the cyanobacterium Nostoc sp. PCC7120 comprises three GAF domains as a sensory module and a histidine kinase as an effector. GAF1 and GAF3 both bind a bilin, and GAF2 contains a tongue-like element. We studied the response of All2699, GAF1-GAF2, and GAF1 to red light by Fourier transform infrared difference spectroscopy, including a 13C-labeled protein moiety for assignment. In GAF1-GAF2, a refolding of the tongue from ß-sheet to α-helix and an upshift of the ring D carbonyl stretch from 1700 to 1712 cm-1 were observed. Therefore, GAF1-GAF2 is regarded as the smallest model system available to study the tongue response and interaction with the chromophore. Replacement of an arginine in the tongue with proline (R387P) did not affect the unfolding of the ß-sheet to Pfr but strongly impaired α-helix formation. In contrast, the Y55H mutation close to bilin ring D did not interfere with conversion to Pfr. Strikingly, the presence of GAF3 in the full-length All2699 diminished the response of the tongue and generated the signal pattern found for GAF1 alone. These results point to a regulatory or integrative role of GAF3 in All2699 that is absent in canonical phytochromes.

The first molecular characterisation of blue- and red-light photoreceptors from Methylobacterium radiotolerans.

Methylobacteria are facultative methylotrophic phytosymbionts of great industrial and agronomical interest, and they are considered as opportunistic pathogens posing a health threat to humans. So far only a few reports mention photoreceptor coding sequences in Methylobacteria genomes, but no investigation at the molecular level has been performed yet. We here present comprehensive in silico research into potential photoreceptors in this bacterial phylum and report the photophysical and photochemical characterisation of two representatives of the most widespread photoreceptor classes, a blue-light sensing LOV (light, oxygen, voltage) protein and a red/far red light sensing BphP (biliverdin-binding bacterial phytochrome) from M. radiotolerans JCM 2831. Overall, both proteins undergo the expected light-triggered reactions, but peculiar features were also identified. The LOV protein Mr4511 has an extremely long photocycle and lacks a tryptophan conserved in ca. 75% of LOV domains. Mutation I37V accelerates the photocycle by one order of magnitude, while the Q112W change underscores the ability of tryptophan in this position to perform efficient energy transfer to the flavin chromophore. Time-resolved photoacoustic experiments showed that Mr4511 has a higher triplet quantum yield than other LOV domains and that the formation of the photoproduct results in a volume expansion, in sharp contrast to other LOV proteins. Mr4511 was found to be astonishingly resistant to denaturation by urea, still showing light-triggered reactions after incubation in urea for more than 20 h. The phytochrome MrBphP1 exhibits the so far most red-shifted absorption maxima for its Pr- and Pfr forms (λmax = 707 nm and 764 nm for the Pr and Pfr forms). The light-driven conversions in both directions occur with relatively high quantum yields of 0.2. Transient ns absorption spectroscopy (µs-ms time range) identifies the decay of the instantaneously formed lumi-intermediate, followed by only one additional intermediate before the formation of the respective final photoproducts for Pr-to-Pfr or Pfr-to-Pr photoconversion, in contrast to other BphPs. The relatively simple photoconversion patterns suggest the absence of the shunt pathways reported for other bacterial phytochromes.

The Lumi-R Intermediates of Prototypical Phytochromes.

Phytochromes are photoreceptors that upon light absorption initiate a physiological reaction cascade. The starting point is the photoisomerization of the tetrapyrrole cofactor in the parent Pr state, followed by thermal relaxation steps culminating in activation of the physiological signal. Here we have employed resonance Raman (RR) spectroscopy to study the chromophore structure in the primary photoproduct Lumi-R, trapped between 130 and 200 K. The investigations covered phytochromes from plants (phyA) and prokaryotes (Cph1, Agp1, CphB, and RpBphP2) including phytochromobilin (PΦB), phycocyanobilin (PCB), and biliverdin (BV). In PΦB- and PCB-binding phyA and Cph1, two Lumi-R states (Lumi-R1, Lumi-R2) were identified and discussed in terms of sequential and parallel reaction models. In Lumi-R1, the chromophore structural changes are restricted to the C-D methine bridge isomerization site but extended throughout the chromophore in Lumi-R2. Formation and decay kinetics as well as photochemical activity depend on the specific protein-chromophore interactions and thus account for the different distribution between Lumi-R1 and Lumi-R2 in the photostationary mixtures of the various PΦB(PCB)-binding phytochromes. For BV-binding bacteriophytochromes, only a single Lumi-R(BV) state was found. In this state, which is similar for Agp1, CphB, and RpBphP2, the chromophore structural changes comprise major torsions of the C-D methine bridge but also perturbations at the A-B methine bridge remote from the isomerization site. The different structures of the photoproducts in PΦB(PCB)-binding phytochromes and BV-binding bacteriophytochromes are attributed to the different disposition of ring D upon isomerization, which leads to distinct protein-chromophore interactions in the Lumi-R states of these two classes of phytochromes.

Structural elements regulating the photochromicity in a cyanobacteriochrome.

The three-dimensional (3D) crystal structures of the GAF3 domain of cyanobacteriochrome Slr1393 (Synechocystis PCC6803) carrying a phycocyanobilin chromophore could be solved in both 15-Z dark-adapted state, Pr, λmax = 649 nm, and 15-E photoproduct, Pg, λmax = 536 nm (resolution, 1.6 and 1.86 Å, respectively). The structural data allowed identifying the large spectral shift of the Pr-to-Pg conversion as resulting from an out-of-plane rotation of the chromophore's peripheral rings and an outward movement of a short helix formed from a formerly unstructured loop. In addition, a third structure (2.1-Å resolution) starting from the photoproduct crystals allowed identification of elements that regulate the absorption maxima. In this peculiar form, generated during X-ray exposition, protein and chromophore conformation still resemble the photoproduct state, except for the D-ring already in 15-Z configuration and tilted out of plane akin the dark state. Due to its formation from the photoproduct, it might be considered an early conformational change initiating the parental state-recovering photocycle. The high quality and the distinct features of the three forms allowed for applying quantum-chemical calculations in the framework of multiscale modeling to rationalize the absorption maxima changes. A systematic analysis of the PCB chromophore in the presence and absence of the protein environment showed that the direct electrostatic effect is negligible on the spectral tuning. However, the protein forces the outer pyrrole rings of the chromophore to deviate from coplanarity, which is identified as the dominating factor for the color regulation.

Dynamics and efficiency of photoswitching in biliverdin-binding phytochromes.

The light-driven conversions between the dark-adapted and the photoproduct state were recorded for bacteriophytochromes (BphP) carrying biliverdin IXα (BV) as chromophore by time-resolved absorption spectroscopy. BphPs can be photoswitched between a red absorbing (Pr, maximum at ca. 700 nm) and a far-red/near-infrared (Pfr, maximum at ca. 750 nm) absorbing state, thereby showing a considerable red-shift with respect to plant phytochromes. Representatives for BphPs studied here are: PstBphP1 from Pseudomonas syringae pv. tomato, for which Pfr is the photoproduct; the bathy-phytochrome PaBphP from Pseudomonas aeruginosa for which instead Pfr is the thermally stable parental state. The third BphP-like protein was FphA from the fungus Aspergillus nidulans, a eukaryotic protein also carrying BV as a chromophore, for which Pr is considered to be the dark-adapted state. All three BphPs show a canonical modular arrangement with a three-domain photosensory module (PAS-GAF-PHY) and a histidine-kinase (HK) signalling domain. The quantum yields for Pr-to-Pfr photoconversion are in the range 0.02-0.12, and 0.04-0.08 for the Pfr-to-Pr route. Photoproducts of both bacterial phytochromes thermally recovered in the dark, whereas for the fungal protein (FphA) both Pr and Pfr forms are thermally stable for days and could be interconverted only by selective irradiation. The photoinduced reactions of all three BV-phytochromes are in general kinetically less complex than those of plant phytochromes, with the notable exception of the Pr-to-Pfr route for PstBphP1. By contrast in the Pfr-to-Pr conversion of FphAN753 the final product is already formed during the very early steps of the process, without formation of any further intermediates: to our knowledge it is the first phytochrome showing this behavior. All three proteins investigated are weakly fluorescent in the Pr form, with a maximum fluorescence quantum yield of 0.02 (PaBphP), and have undetectable fluorescence in the Pfr state.

MAS NMR on a Red/Far-Red Photochromic Cyanobacteriochrome All2699 from Nostoc.

Unlike canonical phytochromes, the GAF domain of cyanobacteriochromes (CBCRs) can bind bilins autonomously and is sufficient for functional photocycles. Despite the astonishing spectral diversity of CBCRs, the GAF1 domain of the three-GAF-domain photoreceptor all2699 from the cyanobacterium Nostoc 7120 is the only CBCR-GAF known that converts from a red-absorbing (Pr) dark state to a far-red-absorbing (Pfr) photoproduct, analogous to the more conservative phytochromes. Here we report a solid-state NMR spectroscopic study of all2699g1 in its Pr state. Conclusive NMR evidence unveils a particular stereochemical heterogeneity at the tetrahedral C31 atom, whereas the crystal structure shows exclusively the R-stereochemistry at this chiral center. Additional NMR experiments were performed on a construct comprising the GAF1 and GAF2 domains of all2699, showing a greater precision in the chromophore-protein interactions in the GAF1-2 construct. A 3D Pr structural model of the all2699g1-2 construct predicts a tongue-like region extending from the GAF2 domain (akin to canonical phytochromes) in the direction of the chromophore, shielding it from the solvent. In addition, this stabilizing element allows exclusively the R-stereochemistry for the chromophore-protein linkage. Site-directed mutagenesis performed on three conserved motifs in the hairpin-like tip confirms the interaction of the tongue region with the GAF1-bound chromophore.