RESUMO
Bacteriophytochromes are light-sensing biological machines that switch between two photoreversible states, Pr and Pfr. Their relative stability is opposite in canonical and bathy bacteriophytochromes, but in both cases the switch between them is triggered by the photoisomerization of an embedded bilin chromophore. We applied an integrated multiscale strategy of excited-state QM/MM nonadiabatic dynamics and (QM/)MM molecular dynamics simulations with enhanced sampling techniques to the Agrobacterium fabrum bathy phytochrome and compared the results with those obtained for the canonical phytochrome Deinococcus radiodurans. Contrary to what recently suggested, we found that photoactivation in both phytochromes is triggered by the same hula-twist motion of the bilin chromophore. However, only in the bathy phytochrome, the bilin reaches the final rotated structure already in the first intermediate. This allows a reorientation of the binding pocket in a microsecond time scale, which can propagate through the entire protein causing the spine to tilt.
Assuntos
Agrobacterium , Deinococcus , Simulação de Dinâmica Molecular , Fitocromo , Fitocromo/química , Fitocromo/metabolismo , Deinococcus/química , Agrobacterium/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Teoria QuânticaRESUMO
Phytochromes are red-light photoreceptors discovered in plants with homologs in bacteria and fungi that regulate a variety of physiological responses. They display a reversible photocycle between two distinct states: a red-light-absorbing Pr state and a far-red light-absorbing Pfr state. The photoconversion regulates the activity of an enzymatic domain, usually a histidine kinase (HK). The molecular mechanism that explains how light controls the HK activity is not understood because structures of unmodified bacterial phytochromes with HK activity are missing. Here, we report three cryo-electron microscopy structures of a wild-type bacterial phytochrome with HK activity determined as Pr and Pfr homodimers and as a Pr/Pfr heterodimer with individual subunits in distinct states. We propose that the Pr/Pfr heterodimer is a physiologically relevant signal transduction intermediate. Our results offer insight into the molecular mechanism that controls the enzymatic activity of the HK as part of a bacterial two-component system that perceives and transduces light signals.
Assuntos
Microscopia Crioeletrônica , Fitocromo , Transdução de Sinais , Fitocromo/metabolismo , Fitocromo/química , Microscopia Crioeletrônica/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Histidina Quinase/metabolismo , Histidina Quinase/química , Modelos Moleculares , Multimerização Proteica , Luz , Fotorreceptores Microbianos/metabolismo , Fotorreceptores Microbianos/químicaRESUMO
Phytochromes (Phys) are a divergent cohort of bili-proteins that detect light through reversible interconversion between dark-adapted Pr and photoactivated Pfr states. While our understandings of downstream events are emerging, it remains unclear how Phys translate light into an interpretable conformational signal. Here, we present models of both states for a dimeric Phy with histidine kinase (HK) activity from the proteobacterium Pseudomonas syringae, which were built from high-resolution cryo-EM maps (2.8-3.4-Å) of the photosensory module (PSM) and its following signaling (S) helix together with lower resolution maps for the downstream output region augmented by RoseTTAFold and AlphaFold structural predictions. The head-to-head models reveal the PSM and its photointerconversion mechanism with strong clarity, while the HK region is interpretable but relatively mobile. Pr/Pfr comparisons show that bilin phototransformation alters PSM architecture culminating in a scissoring motion of the paired S-helices linking the PSMs to the HK bidomains that ends in reorientation of the paired catalytic ATPase modules relative to the phosphoacceptor histidines. This action apparently primes autophosphorylation enroute to phosphotransfer to the cognate DNA-binding response regulator AlgB which drives quorum-sensing behavior through transient association with the photoreceptor. Collectively, these models illustrate how light absorption conformationally translates into accelerated signaling by Phy-type kinases.
Assuntos
Proteínas de Bactérias , Histidina Quinase , Fitocromo , Pseudomonas syringae , Transdução de Sinais , Histidina Quinase/metabolismo , Histidina Quinase/química , Histidina Quinase/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fitocromo/metabolismo , Fitocromo/química , Pseudomonas syringae/metabolismo , Modelos Moleculares , Microscopia Crioeletrônica , Conformação Proteica , Multimerização Proteica , Fotorreceptores Microbianos/metabolismo , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , LuzRESUMO
The excited state processes of a bacteriophytochrome are studied using high-level multireference methods. The various non-radiative channels of deactivation are identified for the chromophore. The effects of the protein environment and substituents are elucidated for these excited state processes. It is observed that while the excited states are completely delocalized in the Franck-Condon (FC) region, they acquire significant charge transfer character near the conical intersections. Earlier studies have emphasized the delocalized nature of the excited states in the FC region, which leads to absorption spectra with minimal Stokes shift [Rumyantsev et al., Sci. Rep., 2015, 5, 18348]. The effect of the protein environment on the vertical excitation energies was minimal, while that on the conical intersection (CI) energetics was significant. This may lead one to believe that it is charge transfer driven. However, energy decomposition analysis shows that it is the effect of the dispersion of nearby residues and the steric effect on the rings and substituents that lead to the large effect of proteins on the energetics of the CIs.
Assuntos
Proteínas de Bactérias , Proteínas de Bactérias/química , Teoria Quântica , Fitocromo/químicaRESUMO
Red and far-red light-sensing phytochromes are widespread in nature, occurring in plants, algae, fungi, and prokaryotes. Despite at least a billion years of evolution, their photosensory modules remain structurally and functionally similar. Conversely, nature has found remarkably different ways of transmitting light signals from the photosensor to diverse physiological responses. We summarize key features of phytochrome structure and function and discuss how these are correlated, from how the bilin environment affects the chromophore to how light induces cellular signals. Recent advances in the structural characterization of bacterial and plant phytochromes have resulted in paradigm changes in phytochrome research that we discuss in the context of present-day knowledge. Finally, we highlight questions that remain to be answered and suggest some of the benefits of understanding phytochrome structure and function.
Assuntos
Fitocromo , Fitocromo/química , Fitocromo/metabolismo , Fitocromo/fisiologia , Plantas/metabolismo , Plantas/química , LuzRESUMO
Cyanobacteriochromes (CBCRs) are distinctive tetrapyrrole (bilin)-binding photoreceptors exclusively found in cyanobacteria. Unlike canonical phytochromes, CBCRs require only a GAF (cGMP-phosphodiesterase/adenylate cyclase/FhlA) domain for autolyase activity to form a bilin adduct via a Cys residue and cis-trans photoisomerization. Apart from the canonical Cys, which attaches covalently to C31 in the A-ring of the bilin, some GAF domains of CBCRs contain a second-Cys in the Asp-Xaa-Cys-Phe (DXCF) motif, responsible for isomerization of phycocyanobilin (PCB) to phycoviolobilin (PVB) and/or for the formation of a reversible 2nd thioether linkage to the C10. Unlike green/teal-absorbing GAF proteins lacking ligation activity, the second-Cys in another teal-absorbing lineage (DXCF blue/teal group) exhibits both isomerization and ligation activity due to the presence of the Tyr instead of His next to the canonical Cys. Herein, we discovered an atypical CBCR GAF protein, Tpl7205g1, belonging to the DXCF blue/teal group, but having His instead of Tyr next to the first-Cys. Consistent with its subfamily, the second-Cys of Tpl7205g1 did not form a thioether linkage at C10 of PCB, showing only isomerization activity. Instead of forming 2nd thioether linkage, this novel GAF protein exhibits a pH-dependent photocycle between protonated 15Z and deprotonated 15E. Site-directed mutagenesis to the GAF scaffolds revealed its combined characteristics, including properties of teal-DXCF CBCRs and red/green-absorbing CBCRs (XRG CBCRs), suggesting itself as the evolutionary bridge between the two CBCR groups. Our study thus sheds light on the expanded spectral tuning characteristics of teal-light absorbing CBCRs and enhances feasibility of engineering these photoreceptors.
Assuntos
Proteínas de Bactérias , Cianobactérias , Optogenética , Fotorreceptores Microbianos , Fitocromo , Fitocromo/química , Fitocromo/metabolismo , Fitocromo/genética , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Cianobactérias/metabolismo , Cianobactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Optogenética/métodos , Luz , Ficocianina/química , Ficocianina/metabolismo , Engenharia de Proteínas/métodos , Ficobilinas/química , Ficobilinas/metabolismo , Sequência de AminoácidosRESUMO
Certain cyanobacteria alter their photosynthetic light absorption between green and red, a phenomenon called complementary chromatic acclimation. The acclimation is regulated by a cyanobacteriochrome-class photosensor that reversibly photoconverts between green-absorbing (Pg) and red-absorbing (Pr) states. Here, we elucidated the structural basis of the green/red photocycle. In the Pg state, the bilin chromophore adopted the extended C15-Z,anti structure within a hydrophobic pocket. Upon photoconversion to the Pr state, the bilin is isomerized to the cyclic C15-E,syn structure, forming a water channel in the pocket. The solvation/desolvation of the bilin causes changes in the protonation state and the stability of π-conjugation at the B ring, leading to a large absorption shift. These results advance our understanding of the enormous spectral diversity of the phytochrome superfamily.
Assuntos
Luz , Cianobactérias/metabolismo , Cianobactérias/fisiologia , Aclimatação , Fotossíntese , Fitocromo/metabolismo , Fitocromo/química , Modelos Moleculares , Pigmentos Biliares/metabolismo , Pigmentos Biliares/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Luz VermelhaRESUMO
Phytochromes (Phys) are a diverse collection of photoreceptors that regulate numerous physiological and developmental processes in microorganisms and plants through photointerconversion between red-light-absorbing Pr and far-red light-absorbing Pfr states. Light is detected by an N-terminal photo-sensing module (PSM) sequentially comprised of Period/ARNT/Sim (PAS), cGMP-phosphodiesterase/adenylyl cyclase/FhlA (GAF), and Phy-specific (PHY) domains, with the bilin chromophore covalently-bound within the GAF domain. Phys sense light via the Pr/Pfr ratio measured by the light-induced rotation of the bilin D-pyrrole ring that triggers conformational changes within the PSM, which for microbial Phys reaches into an output region. A key step is a ß-stranded to α-helical reconfiguration of a hairpin loop extending from the PHY domain to contact the GAF domain. Besides canonical Phys, cyanobacteria express several variants, including a PAS-less subfamily that harbors just the GAF and PHY domains for light detection. Prior 2D-NMR studies of a model PAS-less Phy from Synechococcus_sp._JA-2-3B'a(2-13) (SyB-Cph1) proposed a unique photoconversion mechanism involving an A-pyrrole ring rotation while magic-angle-spinning NMR probing the chromophore proposed the prototypic D-ring flip. To help solve this conundrum, we determined the crystallographic structure of the GAF-PHY region from SyB-Cph1 as Pr. Surprisingly, this structure differs from canonical Phys by having a Pr ZZZsyn,syn,anti bilin configuration but shifted to the activated position in the binding pocket with consequent folding of the hairpin loop to α-helical, an architecture common for Pfr. Collectively, the PSM of SyB-Cph1 as Pr displayed a mix of dark-adapted and photoactivated features whose co-planar A-C pyrrole rings support a D-ring flip mechanism.
Assuntos
Proteínas de Bactérias , Fitocromo , Fitocromo/química , Fitocromo/metabolismo , Fitocromo/genética , Cristalografia por Raios X , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Cianobactérias/metabolismo , Luz , Domínios Proteicos , Modelos Moleculares , Conformação ProteicaRESUMO
Bacteriophytochrome is a photoreceptor protein that contains the biliverdin (BV) chromophore as its active component. The spectra of BV upon mutation remain remarkably unchanged, as far as spectral positions are concerned. This points toward the minimal effect of electrostatic effects on the electronic structure of the chromophore. However, the relative intensities of the Q and Soret bands of the chromophore change dramatically upon mutation. In this work, we delve into the molecular origin of this unusual intensity modulation. Using extensive classical MD and QM/MM calculations, we show that due to mutation, the conformational population of the chromophore changes significantly. The noncovalent interactions, especially the stacking interactions, lead to extra stabilization of the cyclic form in the D207H mutated species as opposed to the open form in the wild-type BV. Thus, unlike the commonly observed direct electrostatic effect on the spectral shift, in the case of BV the difference observed is in varying intensities, and this in turn is driven by a conformational shift due to enhanced stacking interaction.
Assuntos
Fitocromo , Fitocromo/química , Biliverdina/química , Conformação Molecular , Proteínas de Bactérias/químicaRESUMO
As plant photoreceptors, phytochromes are capable of detecting red light and far-red light, thereby governing plant growth. All2699 is a photoreceptor found in Nostoc sp. PCC7120 that specifically responds to red light and far-red light. All2699g1g2 is a truncated protein carrying the first and second GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains of All2699. In this study, we found that, upon exposure to red light, the protein underwent aggregation, resulting in the formation of protein aggregates. Conversely, under far-red light irradiation, these protein aggregates dissociated. We delved into the factors that impact the aggregation of All2699g1g2, focusing on the protein structure. Our findings showed that the GAF2 domain contains a low-complexity (LC) loop region, which plays a crucial role in mediating protein aggregation. Specifically, phenylalanine at position 239 within the LC loop region was identified as a key site for the aggregation process. Furthermore, our research revealed that various factors, including irradiation time, temperature, concentration, NaCl concentration, and pH value, can impact the aggregation of All2699g1g2. The aggregation led to variations in Pfr concentration depending on temperature, NaCl concentration, and pH value. In contrast, ΔLC did not aggregate and therefore lacked responses to these factors. Consequently, the LC loop region of All2699g1g2 extended and enhanced sensory properties.
Assuntos
Proteínas de Bactérias , Luz , Nostoc , Nostoc/metabolismo , Nostoc/química , Nostoc/efeitos da radiação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínios Proteicos , Agregados Proteicos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Pigmentos Biliares/química , Pigmentos Biliares/metabolismo , Concentração de Íons de Hidrogênio , Fitocromo/química , Fitocromo/metabolismoRESUMO
Bathy phytochromes are a subclass of bacterial biliprotein photoreceptors that carry a biliverdin IXα chromophore. In contrast to prototypical phytochromes that adopt a red-light-absorbing Pr ground state, the far-red light-absorbing Pfr-form is the thermally stable ground state of bathy phytochromes. Although the photobiology of bacterial phytochromes has been extensively studied since their discovery in the late 1990s, our understanding of the signal transduction process to the connected transmitter domains, which are often histidine kinases, remains insufficient. Initiated by the analysis of the bathy phytochrome PaBphP from Pseudomonas aeruginosa, we performed a systematic analysis of five different bathy phytochromes with the aim to derive a general statement on the correlation of photostate and autokinase output. While all proteins adopt different Pr/Pfr-fractions in response to red, blue, and far-red light, only darkness leads to a pure or highly enriched Pfr-form, directly correlated with the lowest level of autokinase activity. Using this information, we developed a method to quantitatively correlate the autokinase activity of phytochrome samples with well-defined stationary Pr/Pfr-fractions. We demonstrate that the off-state of the phytochromes is the Pfr-form and that different Pr/Pfr-fractions enable the organisms to fine-tune their kinase output in response to a certain light environment. Furthermore, the output response is regulated by the rate of dark reversion, which differs significantly from 5 s to 50 min half-life. Overall, our study indicates that bathy phytochromes function as sensors of light and darkness, rather than red and far-red light, as originally postulated.
Assuntos
Proteínas de Bactérias , Escuridão , Fitocromo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Histidina Quinase/metabolismo , Histidina Quinase/genética , Luz , Fotorreceptores Microbianos/metabolismo , Fitocromo/metabolismo , Fitocromo/química , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Ativação EnzimáticaRESUMO
Sensor-effector proteins integrate information from different stimuli and transform this into cellular responses. Some sensory domains, like red-light responsive bacteriophytochromes, show remarkable modularity regulating a variety of effectors. One effector domain is the GGDEF diguanylate cyclase catalyzing the formation of the bacterial second messenger cyclic-dimeric-guanosine monophosphate. While critical signal integration elements have been described for different phytochromes, a generalized understanding of signal processing and communication over large distances, roughly 100 Å in phytochrome diguanylate cyclases, is missing. Here we show that dynamics-driven allostery is key to understanding signal integration on a molecular level. We generated protein variants stabilized in their far-red-absorbing Pfr state and demonstrated by analysis of conformational dynamics using hydrogen-deuterium exchange coupled to mass spectrometry that single amino acid replacements are accompanied by altered dynamics of functional elements throughout the protein. We show that the conformational dynamics correlate with the enzymatic activity of these variants, explaining also the increased activity of a non-photochromic variant. In addition, we demonstrate the functional importance of mixed Pfr/intermediate state dimers using a fast-reverting variant that still enables wild-type-like fold-changes of enzymatic stimulation by red light. This supports the functional role of single protomer activation in phytochromes, a property that might correlate with the non-canonical mixed Pfr/intermediate-state spectra observed for many phytochrome systems. We anticipate our results to stimulate research in the direction of dynamics-driven allosteric regulation of different bacteriophytochrome-based sensor-effectors. This will eventually impact design strategies for the creation of novel sensor-effector systems for enriching the optogenetic toolbox.
Assuntos
Luz , Fósforo-Oxigênio Liases , Fitocromo , Regulação Alostérica , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fósforo-Oxigênio Liases/metabolismo , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/genética , Fitocromo/metabolismo , Fitocromo/química , Fitocromo/genética , Multimerização Proteica , Luz Vermelha , Alteromonadaceae/enzimologia , Modelos MolecularesRESUMO
Light and temperature sensing are important features of many organisms. Light may provide energy but may also be used by non-photosynthetic organisms for orientation in the environment. Recent evidence suggests that plant and fungal phytochrome and plant phototropin serve dual functions as light and temperature sensors. Here we characterized the fungal LOV-domain blue-light receptor LreA of Alternaria alternata and show that it predominantly contains FAD as chromophore. Blue-light illumination induced ROS production followed by protein agglomeration in vitro. In vivo ROS may control LreA activity. LreA acts as a blue-light photoreceptor but also triggers temperature-shift-induced gene expression. Both responses required the conserved amino acid cysteine 421. We therefore propose that temperature mimics the photoresponse, which could be the ancient function of the chromoprotein. Temperature-dependent gene expression control with LreA was distinct from the response with phytochrome suggesting fine-tuned, photoreceptor-specific gene regulation.
Assuntos
Alternaria , Luz Azul , Flavina-Adenina Dinucleotídeo , Proteínas Fúngicas , Fotorreceptores Microbianos , Alternaria/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavina-Adenina Dinucleotídeo/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica , Fotorreceptores Microbianos/metabolismo , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Fitocromo/metabolismo , Fitocromo/química , Fitocromo/genética , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismo , TemperaturaRESUMO
Sorghum, a short-day tropical plant, has been adapted for temperate grain production, in particular through the selection of variants at the MATURITY loci (Ma1-Ma6) that reduce photoperiod sensitivity. Ma3 encodes phytochrome B (phyB), a red/far-red photochromic biliprotein photoreceptor. The multi-domain gene product, comprising 1178 amino acids, autocatalytically binds the phytochromobilin chromophore to form the photoactive holophytochrome (Sb.phyB). This study describes the development of an efficient heterologous overproduction system which allows the production of large quantities of various holoprotein constructs, along with purification and crystallization procedures. Crystals of the Pr (red-light-absorbing) forms of NPGP, PGP and PG (residues 1-655, 114-655 and 114-458, respectively), each C-terminally tagged with His6, were successfully produced. While NPGP crystals did not diffract, those of PGP and PG diffracted to 6 and 2.1â Å resolution, respectively. Moving the tag to the N-terminus and replacing phytochromobilin with phycocyanobilin as the ligand produced PG crystals that diffracted to 1.8â Å resolution. These results demonstrate that the diffraction quality of challenging protein crystals can be improved by removing flexible regions, shifting fusion tags and altering small-molecule ligands.
Assuntos
Fitocromo , Sorghum , Fitocromo B/genética , Sorghum/genética , Sorghum/metabolismo , Cristalização , Cristalografia por Raios X , Fitocromo/química , Fitocromo/genética , Fitocromo/metabolismo , LuzRESUMO
Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to the phytochromes sensing red and far-red light reversibly. Only the cGMP phosphodiesterase/Adenylate cyclase/FhlA (GAF) domain is needed for chromophore incorporation and proper photoconversion. The CBCR GAF domains covalently ligate linear tetrapyrrole chromophores and show reversible photoconversion between two light-absorbing states. In most cases, the two light-absorbing states are stable under dark conditions, but in some cases, the photoproduct state undergoes thermal relaxation back to the dark-adapted state during thermal relaxation. In this study, we examined the engineered CBCR GAF domain, AnPixJg2_BV4. AnPixJg2_BV4 covalently binds biliverdin IX-alpha (BV) and shows reversible photoconversion between a far-red-absorbing Pfr dark-adapted state and an orange-absorbing Po photoproduct state. Because the BV is an intrinsic chromophore of mammalian cells and absorbs far-red light penetrating into deep tissues, BV-binding CBCR molecules are useful for the development of optogenetic and bioimaging tools used in mammals. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis on the Phe319 position. We succeeded in obtaining variant molecules with higher chromophore-binding efficiency and higher molar extinction coefficient. Furthermore, we observed a wide variation in thermal relaxation kinetics, with an 81-fold difference between the slowest and fastest rates. Both molecules with relatively slow and fast thermal relaxation would be advantageous for optogenetic control.
Assuntos
Proteínas de Bactérias , Biliverdina , Cianobactérias , Fotorreceptores Microbianos , Fitocromo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biliverdina/química , Cianobactérias/metabolismo , Luz , Mutagênese , Fitocromo/química , Conformação Proteica , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Ligação Proteica , Fenilalanina/química , Fenilalanina/genética , Simulação de Dinâmica MolecularRESUMO
The phytochrome superfamily comprises three groups of photoreceptors sharing a conserved GAF (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA) domain that uses a covalently attached linear tetrapyrrole (bilin) chromophore to sense light. Knotted red/far-red phytochromes are widespread in both bacteria and eukaryotes, but cyanobacteria also contain knotless red/far-red phytochromes and cyanobacteriochromes (CBCRs). Unlike typical phytochromes, CBCRs require only the GAF domain for bilin binding, chromophore ligation, and full, reversible photoconversion. CBCRs can sense a wide range of wavelengths (ca. 330-750 nm) and can regulate phototaxis, second messenger metabolism, and optimization of the cyanobacterial light-harvesting apparatus. However, the origins of CBCRs are not well understood: we do not know when or why CBCRs evolved, or what selective advantages led to retention of early CBCRs in cyanobacterial genomes. In the current work, we use the increasing availability of genomes and metagenome-assembled-genomes from early-branching cyanobacteria to explore the origins of CBCRs. We reaffirm the earliest branches in CBCR evolution. We also show that early-branching cyanobacteria contain late-branching CBCRs, implicating early appearance of CBCRs during cyanobacterial evolution. Moreover, we show that early-branching CBCRs behave as integrators of light and pH, providing a potential unique function for early CBCRs that led to their retention and subsequent diversification. Our results thus provide new insight into the origins of these diverse cyanobacterial photoreceptors.
Assuntos
Proteínas de Bactérias , Cianobactérias , Fotorreceptores Microbianos , Fitocromo , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Fitocromo/química , Fitocromo/classificação , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/classificação , Motivos de AminoácidosRESUMO
Phytochromes constitute a family of photosensory proteins that are utilized by various organisms to regulate several physiological processes. Phytochromes bind a bilin pigment that switches its isomeric state upon absorption of red or far-red photons, resulting in protein conformational changes that are sensed by the organism. Previously, the ultrafast dynamics in bacterial phytochrome was resolved to atomic resolution by time-resolved serial femtosecond X-ray diffraction (TR-SFX), showing extensive changes in its molecular conformation at 1 picosecond delay time. However, the large excitation fluence of mJ/mm2 used in TR-SFX questions the validity of the observed dynamics. In this work, we present an excitation-dependent ultrafast transient absorption study to test the response of a related bacterial phytochrome to excitation fluence. We observe excitation power-dependent sub-picosecond dynamics, assigned to the population of high-lying excited state Sn through resonantly enhanced two-photon absorption, followed by rapid internal conversion to the low-lying S1 state. Inspection of the long-lived spectrum under high fluence shows that in addition to the primary intermediate Lumi-R, spectroscopic signatures of solvated electrons and ionized chromophore radicals are observed. Supported by numerical modelling, we propose that under excitation fluences of tens of µJ/mm2 and higher, bacterial phytochrome partly undergoes photoionization from the Sn state in competition with internal conversion to the S1 state in 300 fs. We suggest that the extensive structural changes of related, shorter bacterial phytochrome, lacking the PHY domain, resolved from TR-SFX may have been affected by the ionized species. We propose approaches to minimize the two-photon absorption process by tuning the excitation spectrum away from the S1 absorption or using phytochromes exhibiting minimized or shifted S1 absorption.
Assuntos
Proteínas de Bactérias , Fitocromo , Proteínas de Bactérias/química , Pigmentos Biliares/química , Isomerismo , Fitocromo/química , Análise Espectral , Absorção Fisico-Química , Conformação Proteica , Difração de Raios XRESUMO
The cyanobacteriochrome Slr1393 can be photoconverted between a red (Pr) and green absorbing form (Pg). The recently determined crystal structures of both states suggest a major movement of Trp496 from a stacking interaction with ring D of the phycocyanobilin (PCB) chromophore in Pr to a position outside the chromophore pocket in Pg. Here, we investigated the role of this amino acid during photoconversion in solution using engineered protein variants in which Trp496 was substituted by natural and non-natural amino acids. These variants and the native protein were studied by various spectroscopic techniques (UV-vis absorption, fluorescence, IR, NIR and UV resonance Raman) complemented by theoretical approaches. Trp496 is shown to affect the electronic transition of PCB and to be essential for the thermal equilibrium between Pr and an intermediate state O600. However, Trp496 is not required to stabilize the tilted orientation of ring D in Pr, and does not play a role in the secondary structure changes of Slr1393 during the Pr/Pg transition. The present results confirm the re-orientation of Trp496 upon Pr â Pg conversion, but do not provide evidence of a major change in the microenvironment of this residue. Structural models indicate the penetration of water molecules into the chromophore pocket in both Pr and Pg states and thus water-Trp contacts, which can readily account for the subtle spectral changes between Pr and Pg. Thus, we conclude that reorientation of Trp496 during the Pr-to-Pg photoconversion in solution is not associated with a major change in the dielectric environment in the two states.
Assuntos
Proteínas de Bactérias , Fotorreceptores Microbianos , Fitocromo , Synechocystis , Triptofano , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Fitocromo/química , Fitocromo/genética , Triptofano/química , Triptofano/genética , Água/química , Conformação ProteicaRESUMO
Sensory photoreceptors abound in nature and enable organisms to adapt behavior, development, and physiology to environmental light. In optogenetics, photoreceptors allow spatiotemporally precise, reversible, and non-invasive control by light of cellular processes. Notwithstanding the development of numerous optogenetic circuits, an unmet demand exists for efficient systems sensitive to red light, given its superior penetration of biological tissue. Bacteriophytochrome photoreceptors sense the ratio of red and far-red light to regulate the activity of enzymatic effector modules. The recombination of bacteriophytochrome photosensor modules with cyclase effectors underlies photoactivated adenylyl cyclases (PAC) that catalyze the synthesis of the ubiquitous second messenger 3', 5'-cyclic adenosine monophosphate (cAMP). Via homologous exchanges of the photosensor unit, we devised novel PACs, with the variant DmPAC exhibiting 40-fold activation of cyclase activity under red light, thus surpassing previous red-light-responsive PACs. Modifications of the PHY tongue modulated the responses to red and far-red light. Exchanges of the cyclase effector offer an avenue to further enhancing PACs but require optimization of the linker to the photosensor. DmPAC and a derivative for 3', 5'-cyclic guanosine monophosphate allow the manipulation of cyclic-nucleotide-dependent processes in mammalian cells by red light. Taken together, we advance the optogenetic control of second-messenger signaling and provide insight into the signaling and design of bacteriophytochrome receptors.