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Joachim E Schultz - One of the best experts on this subject based on the ideXlab platform.
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Structural and biochemical aspects of tandem GAF Domains.
Handbook of experimental pharmacology, 2020Co-Authors: Joachim E SchultzAbstract:The GAF Domain is a small-molecule-binding-Domain (SMBD) identified in >7400 proteins. However, mostly the ligands are unknown. Here we mainly deal with regulatory N-terminal tandem GAF Domains, GAF-A and GAF-B, of four mammalian phosphodiesterases (PDEs) and of two cyanobacterial adenylyl cyclases (ACs) which bind cyclic nucleotides. These tandem GAFs are preceded by N-terminal sequences of variable lengths and a function of their own. In mammals, GAF Domains are found only in cyclic nucleotide PDEs 2, 5, 6, 10, and 11. cAMP is the ligand for phosphodiesterase 10, cGMP for the others. Two cyanobacterial ACs, CyaB1 and 2, carry regulatory cAMP-binding tandem GAF Domains which are similar in sequence to the mammalian ones. These tandem GAF Domains have a prominent NKFDE motif which contributes to ligand binding in an as yet unknown manner. Contradicting structures (parallel vs. antiparallel) are available for the tandem GAF Domains of PDE 2 and AC CyaB2. In addition, the structures of phosphodiesterase 5 and 10 GAF monomers with bound ligands have been solved. In all instances, cyclic nucleotide binding involves specific protein-ligand interactions within a tightly closed binding pocket and minimal solvent exposure of the ligand. The PDE tandem GAF Domains can functionally substitute for the tandem of the cyanobacterial AC CyaB1; e.g. cGMP-regulation is grafted onto the AC using tandem GAFs from PDEs 2, 5 and 11. Studies of GAF Domain-regulated PDEs are hampered by the identities of regulator and substrate molecules. Using AC CyaB1 as a reporter which uses ATP as a substrate solves this issue and makes the tandem GAF Domains of mammalian PDEs available for detailed kinetic and mechanistic studies. In addition, drugs which potentially act on PDE regulatory Domains may be assayed with such a novel test system.
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the GAF tandem Domain of phosphodiesterase 5 as a potential drug target
Handbook of experimental pharmacology, 2011Co-Authors: Joachim E Schultz, Torsten Dunkern, Elvira Gawlittagorka, Gabriele SorgAbstract:Classic PDE5 inhibitors interact with and block the catalytic site of PDE5. They have been clinically validated for treatment of erectile dysfunction as well as reduction of pulmonary arterial pressure, improvement of exercise capacity, quality of life, and arterial oxygenation in patients with secondary pulmonary hypertension. Minor side effects are visual disturbances, headache, migraine, back pain, and interaction with nitrates (hypotension). Some of those side effects presumably can be ameliorated by improving selectivity and pharmacokinetics; other side effects probably are target related due to inhibition of basic physiological processes. Target related side effects may be bypassed by using PDE5 inhibitors with a different mode of action: PDE5, like PDE2, PDE6, PDE10, and PDE11, is a multiDomain protein with an N-terminal tandem GAF Domain, which in case of PDE5, is allosterically activated by cGMP. Potential inhibitors acting at the PDE5 GAF Domain would be expected to inhibit only pathophysiologically upregulated PDE5 activity, whereas basal activity of PDE5 would remain unaffected.
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Functional chimeras of the phosphodiesterase 5 and 10 tandem GAF Domains.
Journal of Biological Chemistry, 2008Co-Authors: Karina Hofbauer, Anita Schultz, Joachim E SchultzAbstract:Abstract The tandem GAF Domain of hPDE10A uses cAMP as an allosteric ligand (Gross-Langenhoff, M., Hofbauer, K., Weber, J., Schultz, A., and Schultz, J. E. (2006) J. Biol. Chem. 281, 2841–2846). We used a two-pronged approach to study how discrimination of ligand is achieved in human (h)PDE10A and how Domain selection in the phosphodiesterase GAF tandems is determined. First, we examined which functional groups of cAMP are responsible for purine ring discrimination. Changes at the C-6 ring position (removal of the amino group; chloride substitution) and at the N-1 ring position reduced stimulation efficacy by 80%, i.e. marking those positions as decisive for nucleotide discrimination. Second, we generated a GAF tandem chimera that consisted of the cGMP-binding GAF-A unit from hPDE5A1, which signals through cGMP in PDE5, and the GAF-B from hPDE10A1, which signals through cAMP in PDE10. Stimulation of the reporter enzyme exclusively was through the GAF-B Domain of hPDE10A1 (EC50 = 7 μm cAMP) as shown by respective point mutations. The PDE5 GAF-A Domain in the chimera did not signal, and its function was reduced to a strictly structural role. Signaling was independent of the origin of the N terminus. Generating 10 additional PDE5/10 tandem GAF chimeras surprisingly demonstrated that the length-conserved linker in GAF tandems between GAF-A and GAF-B played an unforeseen decisive role in intramolecular signaling. Swapping the linker sections between PDE5 and PDE10 GAF tandem Domains abrogated signaling completely pointing to specific Domain interactions within GAF tandems, which are not visible in the available crystal structures with bound ligands.
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the properties of phosphodiesterase 11a4 GAF Domains are regulated by modifications in its n terminal Domain
FEBS Journal, 2008Co-Authors: Marco Grosslangenhoff, Anita Schultz, Arnulf Stenzl, Florian Altenberend, Joachim E SchultzAbstract:The tandem GAF Domain of human phosphodiesterase 11A4 (hPDE11A4) requires 72 μm cGMP for half-maximal effective concentration (EC50) of a cyanobacterial adenylyl cyclase used as a reporter enzyme. Here we examine whether modifications in the N-terminus of PDE11A4 affect cGMP signalling. The N-terminus has two phosphorylation sites for cyclic nucleotide monophosphate-dependent protein kinases (Ser117, Ser168). Phosphorylation of both by cAMP-dependent protein kinase decreased the EC50 value for cGMP from 72 to 23 μm. Phosphomimetic point mutations (S117D/S167D), which project complete phosphorylation, lowered the EC50 value to 16 μm. Structural and sequence data indicate that 196 amino acids precede the start of the GAF Domain in hPDE11A4. Removal of 197 amino acids yielded unregulated cyclase activity, whereas truncation by 196 amino acids resulted in a cGMP-regulated protein with a cGMP EC50 value of 7.6 μm. Truncation by 176 amino acids was required for cGMP EC50 values to decrease to below 10 μm; a construct truncated by 168 amino acids had an EC50 value of 224 μm. The decrease in EC50 values was accompanied by a sixfold increase in basal activity; the extent of cGMP stimulation remained unaffected, however. We conclude that N-terminal modifications strongly affect cGMP regulation of hPDE11A4.
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Characterization of tandem GAF Domains of phosphodiesterases
BMC Pharmacology, 2007Co-Authors: Joachim E Schultz, Karina Hofbauer, Marco Gross-langenhoffAbstract:In mammals, cGMP is generated by soluble and membrane-bound guanylyl cyclases and can be degraded by altogether eight out of 11 mammalian PDE families. Apart from cGMP-activated protein kinases and from cGMPgated cation channels 5 of the 11 PDE families are also subject to regulation by cNMP via their N-terminal tandem GAF ensembles. Four, PDE 2, 5, 6, and 11 appear to be regulated by cGMP; PDE10 is similarly regulated by cAMP. Thus, the cyclic nucleotides act as allosteric activators which enhance their own degradation at the catalytic site, i.e. they concomitantly serve as modulators of enzyme activity and as substrates. This creates a biochemical conundrum which cannot be disentangled kinetically. We use a cyanobacterial adenylyl cyclase, cyaB1, as a reporter enzyme to characterize intramolecular GAF Domain signalling. This cyclase has an N-terminal GAF tandem which is similar to those in mammalian PDEs and regulates cyclase activity in a feed-forward manner using the product cAMP as an activator. Surprisingly, the GAF tandem Domains of PDE 2, 5, 10, and 11 functionally couple to the cyclase and regulate it in a manner consistent with their function in the respective PDEs [1-3].
Masahiko Ikeuchi - One of the best experts on this subject based on the ideXlab platform.
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Distinctive Properties of Dark Reversion Kinetics between Two Red/Green-Type Cyanobacteriochromes and their Application in the Photoregulation of cAMP Synthesis
Photochemistry and Photobiology, 2017Co-Authors: Keiji Fushimi, Masahiko Ikeuchi, Enomoto, Rei NarikawaAbstract:: Cyanobacteriochromes (CBCRs) are photoreceptors that bind to a linear tetrapyrrole within a conserved cGMP-phosphodiesterase/adenylate cyclase/FhlA (GAF) Domain and exhibit reversible photoconversion. Red/green-type CBCR GAF Domains that photoconvert between red- (Pr) and green-absorbing (Pg) forms occur widely in various cyanobacteria. A putative phototaxis regulator, AnPixJ, contains multiple red/green-type CBCR GAF Domains. We previously reported that AnPixJ's second Domain (AnPixJg2) but not its fourth Domain (AnPixJg4) shows red/green reversible photoconversion. Herein, we found that AnPixJg4 showed Pr-to-Pg photoconversion and rapid Pg-to-Pr dark reversion, whereas AnPixJg2 showed a barely detectable dark reversion. Site-directed mutagenesis revealed the involvement of six residues in Pg stability. Replacement at the Leu294/Ile660 positions of AnPixJg2/AnPixJg4 showed the highest influence on dark reversion kinetics. AnPixJg2_DR6, wherein the six residues of AnPixJg2 were entirely replaced with those of AnPixJg4, showed a 300-fold faster dark reversion than that of the wild type. We constructed chimeric proteins by fusing the GAF Domains with adenylate cyclase catalytic regions, such as AnPixJg2-AC, AnPixJg4-AC and AnPixJg2_DR6-AC. We detected successful enzymatic activation under red light for both AnPixJg2-AC and AnPixJg2_DR6-AC, and repression under green light for AnPixJg2-AC and under dark incubation for AnPixJg2_DR6-AC. These results provide platforms to develop cAMP synthetic optogenetic tools.
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a new type of dual cys cyanobacteriochrome GAF Domain found in cyanobacterium acaryochloris marina which has an unusual red blue reversible photoconversion cycle
Biochemistry, 2014Co-Authors: Rei Narikawa, Keiji Fushimi, Gen Enomoto, Masahiko IkeuchiAbstract:Cyanobacteriochromes (CBCRs) form a large, spectrally diverse family of photoreceptors (linear tetrapyrrole covalently bound via a conserved cysteine) that perceive ultraviolet to red light. The underlying mechanisms are reasonably well understood with, in certain cases, reversible formation of an adduct between a second cysteine and the chromophore accounting, in part, for their spectral diversity. These CBCRs are denoted as dual-Cys CBCRs, and most such CBCRs had been shown to reversibly absorb blue and green light. Herein, we report the structural and mechanistic characterization of a new type of dual-Cys CBCR, AM1_1186, which exhibits reversible photoconversion between a red-absorbing dark state (λmax = 641 nm) and a blue-absorbing photoproduct (λmax = 416 nm). The wavelength separation of AM1_1186 photoconversion is the largest found to date for a CBCR. In addition to one well-conserved cysteine responsible for covalent incorporation of the chromophore into the apoprotein, AM1_1186 contains a second ...
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photoconversion mechanism of the second GAF Domain of cyanobacteriochrome anpixj and the cofactor structure of its green absorbing state
Biochemistry, 2013Co-Authors: Francisco Velazquez Escobar, Tillmann Utesch, Rei Narikawa, Masahiko Ikeuchi, Maria Andrea Mroginski, Wolfgang Gartner, Peter HildebrandtAbstract:Cyanobacteriochromes are members of the phytochrome superfamily. In contrast to classical phytochromes, these small photosensors display a considerable variability of electronic absorption maxima. We have studied the light-induced conversions of the second GAF Domain of AnPixJ, AnPixJg2, a phycocyanobilin-binding protein from the cyanobacterium Anabaena PCC 7120, using low-temperature resonance Raman spectroscopy combined with molecular dynamics simulations. AnPixJg2 is formed biosynthetically as a red-absorbing form (Pr) and can be photoconverted into a green-absorbing form (Pg). Forward and backward phototransformations involve the same reaction sequences and intermediates of similar cofactor structures as the corresponding processes in canonical phytochromes, including a transient cofactor deprotonation. Whereas the cofactor of the Pr state shows far-reaching similarities to the Pr states of classical phytochromes, the Pg form displays significant upshifts of the methine bridge stretching frequencies c...
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Structures of cyanobacteriochromes from phototaxis regulators AnPixJ and TePixJ reveal general and specific photoconversion mechanism.
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Rei Narikawa, Takami Ishizuka, Norifumi Muraki, Tomoo Shiba, Genji Kurisu, Masahiko IkeuchiAbstract:Cyanobacteriochromes are cyanobacterial tetrapyrrole-binding photoreceptors that share a bilin-binding GAF Domain with photoreceptors of the phytochrome family. Cyanobacteriochromes are divided into many subclasses with distinct spectral properties. Among them, putative phototaxis regulators PixJs of Anabaena sp. PCC 7120 and Thermosynechococcus elongatus BP-1 (denoted as AnPixJ and TePixJ, respectively) are representative of subclasses showing red-green-type and blue/green-type reversible photoconversion, respectively. Here, we determined crystal structures for the AnPixJ GAF Domain in its red-absorbing 15Z state (Pr) and the TePixJ GAF Domain in its green-absorbing 15E state (Pg). The overall structure of these proteins is similar to each other and also similar to known phytochromes. Critical differences found are as follows: (i) the chromophore of AnPixJ Pr is phycocyanobilin in a C5-Z,syn/C10-Z,syn/C15-Z,anti configuration and that of TePixJ Pg is phycoviolobilin in a C10-Z,syn/C15-E,anti configuration, (ii) a side chain of the key aspartic acid is hydrogen bonded to the tetrapyrrole rings A, B and C in AnPixJ Pr and to the pyrrole ring D in TePixJ Pg, (iii) additional protein-chromophore interactions are provided by subclass-specific residues including tryptophan in AnPixJ and cysteine in TePixJ. Possible structural changes following the photoisomerization of the chromophore between C15-Z and C15-E are discussed based on the X-ray structures at 1.8 and 2.0-A resolution, respectively, in two distinct configurations.
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characterization of the photoactive GAF Domain of the cika homolog sycika slr1969 of the cyanobacteriumsynechocystis sp pcc 6803
Photochemical and Photobiological Sciences, 2008Co-Authors: Rei Narikawa, Takayuki Kohchi, Masahiko IkeuchiAbstract:Phytochromes and bacteriophytochromes in plants and some species of bacteria, respectively, are photoreceptors that bind linear tetrapyrroles and can respond to red and far-red light signals in a reversible manner. A related but distinct photoreceptor candidate, CikA (denoted ScCikA), has been reported to reset the circadian clock in the cyanobacteriumSynechococcus elongatus PCC 7942 after a dark pulse. However, recent studies have indicated that ScCikA does not function as a photoreceptor but as a redox sensor. Moreover, the Cys residue that covalently ligates the chromophore in phytochromes is not conserved in the ScCikA protein. On the other hand, the CikA homolog in Synechocystis sp. PCC 6803 (Slr1969, denoted SyCikA) retains this conserved Cys residue. In our present study, we have isolated the putative chromophore-binding GAF Domain of SyCikA from Synechocystis and phycocyanobilin-producing Escherichia coli.Absorption spectra of both preparations showed two peaks in the UV and violet regions. Irradiation of these proteins with violet light yielded a broad peak in a yellow region at the expense of the peaks in the UV and violet regions. Interestingly, successive irradiation with yellow light did not revert these absorption spectra but a partial dark reversion to the original form was detected. These results suggest that SyCikA may function as a violet light sensor in Synechocystis.
Kaihong Zhao - One of the best experts on this subject based on the ideXlab platform.
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The Red-/Green-Switching GAF3 of Cyanobacteriochrome Slr1393 from Synechocystis sp. PCC6803 Regulates the Activity of an Adenylyl Cyclase.
ChemBioChem, 2018Co-Authors: Ping‐ping Hu, Ming Zhou, Wolfgang Gartner, Kaihong ZhaoAbstract:: Cyanobacteriochromes (CBCRs) are photoreceptors in cyanobacteria that present a bilin chromophore-binding GAF Domain as a photochromic element to control the activity of a downstream enzyme or regulator. CBCR Slr1393 from Synechocystis PCC 6803 carries three GAF Domains, but only the third one binds phycocyanobilin covalently. Slr1393 shows photochromicity between red and green absorbing states and regulates a C-terminally located histidine kinase. In this work, we fused this third GAF Domain to an adenylyl cyclase (AC) from Microcoleus chthonoplastes PCC7420 that in its genuine form is under blue-light control from a LOV Domain. A series of RGS-AC variants were constructed with various lengths of the linkers between RGS and AC. Assays in vitro and in living Escherichia coli cells (AC-deletion mutant) demonstrated that the activity of AC was light regulated, namely, the red-light-converted form of RGSΔ14-Δ4AC (in vitro) was about three times more active than the green-light-converted form. Expression of the fusion protein RGSΔ14-Δ4AC in vivo again showed highest light regulation with at least threefold amplification of the AC function. In some experiments, even tenfold higher activity was observed, which indicated that the protein, if expressed under in vivo conditions, was part of the E. coli physiological conditions and thereby subjected to more complex and variable regulation through other E. coli inherent factors.
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conversion of phycocyanobilin binding GAF Domain to biliverdin binding Domain
Journal of Porphyrins and Phthalocyanines, 2018Co-Authors: Pingping Hu, Suping Jiang, Ming Zhou, Kaihong ZhaoAbstract:Cyanobacteriochromes (CBCRs) are biliprotein photoreceptors that only exist in cyanobacteria and have a broad spectral response range from ultra-violet to far-red. The red/green-type CBCRs can show red/green reversible photoconversion via a covalently bound phycocyanobilin (PCB). In recent years, several CBCRs binding with not only PCB but also biliverdin (BV) have been discovered, which raises the possibility of CBCRs being applied as optogenetic tools. Through molecular modification, we hope to engineer BV-binding CBCRs responsive to the near-infrared spectral region (650–900 nm), of which the red/green type of CBCRs are suitable resources for experimentation. Here, we use Slr1393g3 (the third GAF Domain of a red/green photoswitching CBCR from Synechocystis sp. PCC 6803) as a template to perform such molecular evolution using both random mutagenesis and site-directed mutagenesis. After several rounds of random mutagenesis, we obtained several BV-binding variants of Slr1393g3. These BV-binding variants h...
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Conversion of phycocyanobilin-binding GAF Domain to biliverdin-binding Domain
Journal of Porphyrins and Phthalocyanines, 2018Co-Authors: Pingping Hu, Suping Jiang, Ming Zhou, Kaihong ZhaoAbstract:Cyanobacteriochromes (CBCRs) are biliprotein photoreceptors that only exist in cyanobacteria and have a broad spectral response range from ultra-violet to far-red. The red/green-type CBCRs can show red/green reversible photoconversion via a covalently bound phycocyanobilin (PCB). In recent years, several CBCRs binding with not only PCB but also biliverdin (BV) have been discovered, which raises the possibility of CBCRs being applied as optogenetic tools. Through molecular modification, we hope to engineer BV-binding CBCRs responsive to the near-infrared spectral region (650–900 nm), of which the red/green type of CBCRs are suitable resources for experimentation. Here, we use Slr1393g3 (the third GAF Domain of a red/green photoswitching CBCR from Synechocystis sp. PCC 6803) as a template to perform such molecular evolution using both random mutagenesis and site-directed mutagenesis. After several rounds of random mutagenesis, we obtained several BV-binding variants of Slr1393g3. These BV-binding variants have a maximal absorbance at ̃690 nm and a fluorescence at ̃720 nm. Additionally, some of them have remarkable photochromicity between a far-red light-absorbing state and a red light-absorbing state. Based on the primary amino acid sequence and structural models, the Phe474 surrounding ring D of BV is thought as a crucial site for chromophore selectivity.
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near infrared fluorescent biliproteins generated from bacteriophytochrome aphb of nostoc sp pcc 7120
Photochemical and Photobiological Sciences, 2016Co-Authors: Che Yuan, Ming Zhou, Wolfgang Gartner, Kun Tang, Hugo Scheer, Huizhen Li, Kaihong ZhaoAbstract:The genome of the cyanobacterium Nostoc sp. PCC 7120 encodes a large number of putative bacteriophytochrome and cyanobacteriochrome photoreceptors that, due to their long-wavelength absorption and fluorescence emission, might serve as fluorescent tags in intracellular investigations. We show that the PAS-GAF Domain of the bacteriophytochrome, AphB, binds biliverdin covalently and exhibits, besides its reversible photochemistry, a moderate fluorescence in the near infrared (NIR) spectral region. It was selected for further increasing the brightness while retaining the NIR fluorescence. In the first step, amino acids assumed to improve fluorescence were selectively mutated. The resulting variants were then subjected to several rounds of random mutagenesis and screened for enhanced fluorescence in the NIR. The brightness of optimized PAS-GAF variants increased more than threefold compared to that of wt AphB(1–321), with only insignificant spectral shifts (Amax around 695 nm, and Fmax around 720 nm). In general, the brightness increases with decreasing wavelengths, which allows for a selection of the fluorophore depending on the optical properties of the tissue. A spectral heterogeneity was observed when residue His260, located in close proximity to the chromophore, was mutated to Tyr, emphasizing the strong effects of the environment on the electronic properties of the bound biliverdin chromophore.
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redox dependent ligand switching in a sensory heme binding GAF Domain of the cyanobacterium nostoc sp pcc7120
Journal of Biological Chemistry, 2015Co-Authors: Ming Zhou, Kaihong Zhao, Kun Tang, Markus Knipp, Robert Stabel, Qi He, Hugo Scheer, Wolfgang GartnerAbstract:Abstract The genome of the cyanobacterium Nostoc sp. PCC7120 carries three genes (all4978, all7016, and alr7522) encoding putative heme-binding GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) proteins that were annotated as transcriptional regulators. They are composed of an N-terminal cofactor Domain and a C-terminal helix-turn-helix motif. All4978 showed the highest affinity for protoheme binding. The heme binding capability of All7016 was moderate, and Alr7522 did not bind heme at all. The “as isolated” form of All4978, identified by Soret band (λmax = 427 nm), was assigned by electronic absorption, EPR, and resonance Raman spectroscopy as a hexa-coordinated low spin FeIII heme with a distal cysteine ligand (absorption of δ-band around 360 nm). The protoheme cofactor is noncovalently incorporated. Reduction of the heme could be accomplished by chemically using sodium dithionite and electrospectrochemically; this latter method yielded remarkably low midpoint potentials of −445 and −453 mV (following Soret and α-band absorption changes, respectively). The reduced form of the heme (FeII state) binds both NO and CO. Cysteine coordination of the as isolated FeIII protein is unambiguous, but interestingly, the reduced heme instead displays spectral features indicative of histidine coordination. Cys-His ligand switches have been reported as putative signaling mechanisms in other heme-binding proteins; however, these novel cyanobacterial proteins are the first where such a ligand-switch mechanism has been observed in a GAF Domain. DNA binding of the helix-turn-helix Domain was investigated using a DNA sequence motif from its own promoter region. Formation of a protein-DNA complex preferentially formed in ferric state of the protein.
Kun Tang - One of the best experts on this subject based on the ideXlab platform.
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near infrared fluorescent biliproteins generated from bacteriophytochrome aphb of nostoc sp pcc 7120
Photochemical and Photobiological Sciences, 2016Co-Authors: Che Yuan, Ming Zhou, Wolfgang Gartner, Kun Tang, Hugo Scheer, Huizhen Li, Kaihong ZhaoAbstract:The genome of the cyanobacterium Nostoc sp. PCC 7120 encodes a large number of putative bacteriophytochrome and cyanobacteriochrome photoreceptors that, due to their long-wavelength absorption and fluorescence emission, might serve as fluorescent tags in intracellular investigations. We show that the PAS-GAF Domain of the bacteriophytochrome, AphB, binds biliverdin covalently and exhibits, besides its reversible photochemistry, a moderate fluorescence in the near infrared (NIR) spectral region. It was selected for further increasing the brightness while retaining the NIR fluorescence. In the first step, amino acids assumed to improve fluorescence were selectively mutated. The resulting variants were then subjected to several rounds of random mutagenesis and screened for enhanced fluorescence in the NIR. The brightness of optimized PAS-GAF variants increased more than threefold compared to that of wt AphB(1–321), with only insignificant spectral shifts (Amax around 695 nm, and Fmax around 720 nm). In general, the brightness increases with decreasing wavelengths, which allows for a selection of the fluorophore depending on the optical properties of the tissue. A spectral heterogeneity was observed when residue His260, located in close proximity to the chromophore, was mutated to Tyr, emphasizing the strong effects of the environment on the electronic properties of the bound biliverdin chromophore.
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redox dependent ligand switching in a sensory heme binding GAF Domain of the cyanobacterium nostoc sp pcc7120
Journal of Biological Chemistry, 2015Co-Authors: Ming Zhou, Kaihong Zhao, Kun Tang, Markus Knipp, Robert Stabel, Qi He, Hugo Scheer, Wolfgang GartnerAbstract:Abstract The genome of the cyanobacterium Nostoc sp. PCC7120 carries three genes (all4978, all7016, and alr7522) encoding putative heme-binding GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) proteins that were annotated as transcriptional regulators. They are composed of an N-terminal cofactor Domain and a C-terminal helix-turn-helix motif. All4978 showed the highest affinity for protoheme binding. The heme binding capability of All7016 was moderate, and Alr7522 did not bind heme at all. The “as isolated” form of All4978, identified by Soret band (λmax = 427 nm), was assigned by electronic absorption, EPR, and resonance Raman spectroscopy as a hexa-coordinated low spin FeIII heme with a distal cysteine ligand (absorption of δ-band around 360 nm). The protoheme cofactor is noncovalently incorporated. Reduction of the heme could be accomplished by chemically using sodium dithionite and electrospectrochemically; this latter method yielded remarkably low midpoint potentials of −445 and −453 mV (following Soret and α-band absorption changes, respectively). The reduced form of the heme (FeII state) binds both NO and CO. Cysteine coordination of the as isolated FeIII protein is unambiguous, but interestingly, the reduced heme instead displays spectral features indicative of histidine coordination. Cys-His ligand switches have been reported as putative signaling mechanisms in other heme-binding proteins; however, these novel cyanobacterial proteins are the first where such a ligand-switch mechanism has been observed in a GAF Domain. DNA binding of the helix-turn-helix Domain was investigated using a DNA sequence motif from its own promoter region. Formation of a protein-DNA complex preferentially formed in ferric state of the protein.
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combined mutagenesis and kinetics characterization of the bilin binding GAF Domain of the protein slr1393 from the cyanobacterium synechocystis pcc6803
ChemBioChem, 2014Co-Authors: Xiu Ling Xu, Kun Tang, Alexander Gutt, Jonas Mechelke, Sarah Raffelberg, Dan Miao, Lorena Valle, Claudio D Borsarelli, Kaihong ZhaoAbstract:The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF Domains, a PAS Domain, and a histidine kinase Domain. GAF3 is the sole Domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (lmax = 649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 Domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 ms, 390 ms, and 1.5 ms) for the red-to-green conversion, and 1.2 ms, 340 ms, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the greenabsorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.
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Combined Mutagenesis and Kinetics Characterization of the Bilin‐Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803
ChemBioChem, 2014Co-Authors: Xiu Ling Xu, Kaihong Zhao, Kun Tang, Alexander Gutt, Jonas Mechelke, Sarah Raffelberg, Dan Miao, Lorena Valle, Claudio D Borsarelli, Wolfgang GartnerAbstract:The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF Domains, a PAS Domain, and a histidine kinase Domain. GAF3 is the sole Domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (lmax = 649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 Domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 ms, 390 ms, and 1.5 ms) for the red-to-green conversion, and 1.2 ms, 340 ms, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the greenabsorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.
Wolfgang Gartner - One of the best experts on this subject based on the ideXlab platform.
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The Red-/Green-Switching GAF3 of Cyanobacteriochrome Slr1393 from Synechocystis sp. PCC6803 Regulates the Activity of an Adenylyl Cyclase.
ChemBioChem, 2018Co-Authors: Ping‐ping Hu, Ming Zhou, Wolfgang Gartner, Kaihong ZhaoAbstract:: Cyanobacteriochromes (CBCRs) are photoreceptors in cyanobacteria that present a bilin chromophore-binding GAF Domain as a photochromic element to control the activity of a downstream enzyme or regulator. CBCR Slr1393 from Synechocystis PCC 6803 carries three GAF Domains, but only the third one binds phycocyanobilin covalently. Slr1393 shows photochromicity between red and green absorbing states and regulates a C-terminally located histidine kinase. In this work, we fused this third GAF Domain to an adenylyl cyclase (AC) from Microcoleus chthonoplastes PCC7420 that in its genuine form is under blue-light control from a LOV Domain. A series of RGS-AC variants were constructed with various lengths of the linkers between RGS and AC. Assays in vitro and in living Escherichia coli cells (AC-deletion mutant) demonstrated that the activity of AC was light regulated, namely, the red-light-converted form of RGSΔ14-Δ4AC (in vitro) was about three times more active than the green-light-converted form. Expression of the fusion protein RGSΔ14-Δ4AC in vivo again showed highest light regulation with at least threefold amplification of the AC function. In some experiments, even tenfold higher activity was observed, which indicated that the protein, if expressed under in vivo conditions, was part of the E. coli physiological conditions and thereby subjected to more complex and variable regulation through other E. coli inherent factors.
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near infrared fluorescent biliproteins generated from bacteriophytochrome aphb of nostoc sp pcc 7120
Photochemical and Photobiological Sciences, 2016Co-Authors: Che Yuan, Ming Zhou, Wolfgang Gartner, Kun Tang, Hugo Scheer, Huizhen Li, Kaihong ZhaoAbstract:The genome of the cyanobacterium Nostoc sp. PCC 7120 encodes a large number of putative bacteriophytochrome and cyanobacteriochrome photoreceptors that, due to their long-wavelength absorption and fluorescence emission, might serve as fluorescent tags in intracellular investigations. We show that the PAS-GAF Domain of the bacteriophytochrome, AphB, binds biliverdin covalently and exhibits, besides its reversible photochemistry, a moderate fluorescence in the near infrared (NIR) spectral region. It was selected for further increasing the brightness while retaining the NIR fluorescence. In the first step, amino acids assumed to improve fluorescence were selectively mutated. The resulting variants were then subjected to several rounds of random mutagenesis and screened for enhanced fluorescence in the NIR. The brightness of optimized PAS-GAF variants increased more than threefold compared to that of wt AphB(1–321), with only insignificant spectral shifts (Amax around 695 nm, and Fmax around 720 nm). In general, the brightness increases with decreasing wavelengths, which allows for a selection of the fluorophore depending on the optical properties of the tissue. A spectral heterogeneity was observed when residue His260, located in close proximity to the chromophore, was mutated to Tyr, emphasizing the strong effects of the environment on the electronic properties of the bound biliverdin chromophore.
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redox dependent ligand switching in a sensory heme binding GAF Domain of the cyanobacterium nostoc sp pcc7120
Journal of Biological Chemistry, 2015Co-Authors: Ming Zhou, Kaihong Zhao, Kun Tang, Markus Knipp, Robert Stabel, Qi He, Hugo Scheer, Wolfgang GartnerAbstract:Abstract The genome of the cyanobacterium Nostoc sp. PCC7120 carries three genes (all4978, all7016, and alr7522) encoding putative heme-binding GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) proteins that were annotated as transcriptional regulators. They are composed of an N-terminal cofactor Domain and a C-terminal helix-turn-helix motif. All4978 showed the highest affinity for protoheme binding. The heme binding capability of All7016 was moderate, and Alr7522 did not bind heme at all. The “as isolated” form of All4978, identified by Soret band (λmax = 427 nm), was assigned by electronic absorption, EPR, and resonance Raman spectroscopy as a hexa-coordinated low spin FeIII heme with a distal cysteine ligand (absorption of δ-band around 360 nm). The protoheme cofactor is noncovalently incorporated. Reduction of the heme could be accomplished by chemically using sodium dithionite and electrospectrochemically; this latter method yielded remarkably low midpoint potentials of −445 and −453 mV (following Soret and α-band absorption changes, respectively). The reduced form of the heme (FeII state) binds both NO and CO. Cysteine coordination of the as isolated FeIII protein is unambiguous, but interestingly, the reduced heme instead displays spectral features indicative of histidine coordination. Cys-His ligand switches have been reported as putative signaling mechanisms in other heme-binding proteins; however, these novel cyanobacterial proteins are the first where such a ligand-switch mechanism has been observed in a GAF Domain. DNA binding of the helix-turn-helix Domain was investigated using a DNA sequence motif from its own promoter region. Formation of a protein-DNA complex preferentially formed in ferric state of the protein.
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Combined Mutagenesis and Kinetics Characterization of the Bilin‐Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803
ChemBioChem, 2014Co-Authors: Xiu Ling Xu, Kaihong Zhao, Kun Tang, Alexander Gutt, Jonas Mechelke, Sarah Raffelberg, Dan Miao, Lorena Valle, Claudio D Borsarelli, Wolfgang GartnerAbstract:The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF Domains, a PAS Domain, and a histidine kinase Domain. GAF3 is the sole Domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (lmax = 649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 Domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 ms, 390 ms, and 1.5 ms) for the red-to-green conversion, and 1.2 ms, 340 ms, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the greenabsorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.
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photoconversion mechanism of the second GAF Domain of cyanobacteriochrome anpixj and the cofactor structure of its green absorbing state
Biochemistry, 2013Co-Authors: Francisco Velazquez Escobar, Tillmann Utesch, Rei Narikawa, Masahiko Ikeuchi, Maria Andrea Mroginski, Wolfgang Gartner, Peter HildebrandtAbstract:Cyanobacteriochromes are members of the phytochrome superfamily. In contrast to classical phytochromes, these small photosensors display a considerable variability of electronic absorption maxima. We have studied the light-induced conversions of the second GAF Domain of AnPixJ, AnPixJg2, a phycocyanobilin-binding protein from the cyanobacterium Anabaena PCC 7120, using low-temperature resonance Raman spectroscopy combined with molecular dynamics simulations. AnPixJg2 is formed biosynthetically as a red-absorbing form (Pr) and can be photoconverted into a green-absorbing form (Pg). Forward and backward phototransformations involve the same reaction sequences and intermediates of similar cofactor structures as the corresponding processes in canonical phytochromes, including a transient cofactor deprotonation. Whereas the cofactor of the Pr state shows far-reaching similarities to the Pr states of classical phytochromes, the Pg form displays significant upshifts of the methine bridge stretching frequencies c...
