Dynamic Structural Changes Underpin Photoconversion of a Blue/Green Cyanobacteriochrome between Its Dark and Photoactivated States

The phytochrome superfamily of photoreceptors exploits reversible light-driven changes in the bilin chromophore to initiate a variety of signaling cascades. The nature of these alterations and how they impact the protein moiety remain poorly resolved and might include several species-specific routes. Here, we provide a detailed picture of photoconversion for the photosensing cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain from Thermosynechococcus elongatus (Te) PixJ, a member of the cyanobacteriochrome clade. Solution NMR structures of the blue light-absorbing dark state Pb and green light-absorbing photoactivated state Pg, combined with paired crystallographic models, revealed that the bilin and GAF domain dynamically transition via breakage of the C10/Cys-494 thioether bond, opposite rotations of the A and D pyrrole rings, sliding of the bilin in the GAF pocket, and the appearance of an extended region of disorder that includes Cys-494. Changes in GAF domain backbone dynamics were also observed that are likely important for inter-domain signal propagation. Taken together, photoconversion of T. elongatus PixJ from Pb to Pg involves complex structural changes within the GAF domain pocket that transduce light into a mechanical signal, many aspects of which should be relevant to others within the extended phytochrome superfamily.     

Cyanobacterial phytochrome-like PixJ1 holoprotein shows novel reversible photoconversion between blue- and green-absorbing forms

The gene, pixJ1 (formerly pisJ1), is predicted to encode a phytochrome-like photoreceptor that is essential for positive phototaxis in the unicellular cyanobacterium Synechocystis sp. PCC 6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The PixJ1 protein was overexpressed as a fusion with a poly-histidine tag (His-PixJ1) and isolated from Synechocystis cells. A zinc-fluorescence assay suggested that a linear tetrapyrrole was covalently attached to the His-PixJ1 protein as a chromophore. His-PixJ1 showed novel photoreversible conversion between a blue light-absorbing form (Pb, lambdaAmax=425-435 nm) and a green light-absorbing form (Pg, lambdaAmax=535 nm). Dark incubation led Pg to revert to Pb, indicative of stability of the Pb form in darkness. Red or far-red light irradiation, which is effective for photochemical conversion of the known phytochromes, produced no change in the spectra of Pb and Pg forms. Site-directed mutagenesis revealed that a Cys-His motif in the second GAF domain of PixJ1 is responsible for binding of the chromophore. Possible chromophore species are discussed with regard to the novel photoconversion spectrum.     

Cyanobacteriochrome TePixJ of Thermosynechococcus elongatus harbors phycoviolobilin as a chromophore

Cyanobacteria have several putative photoreceptors (designated cyanobacteriochromes) that are related to but distinct from the established phytochromes. The GAF domain of the phototaxis regulator, PixJ, from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixJ_GAF) is a cyanobacteriochrome which exhibits reversible photoconversion between a blue light-absorbing form (max = 433 nm) and a green light-absorbing form (max = 531 nm). To study the chromophore, we prepared TePixJ_GAF chromoprotein from heterologously expressed Synechocystis and performed spectral analysis after denaturation by comparing it with the cyanobacterial phytochrome Cph1 which harbors phycocyanobilin (PCB) as a chromophore. The results indicated that the chromophore of TePixJ is not PCB, but its isomer, phycoviolobilin (PVB). It is suggested that the GAF domain of TePixJ has auto-lyase and auto-isomerase activities.     

嗜热藻BP-1中几个蓝细菌光敏色素结构域体内重组及光化学性质研究

通过蛋白质序列同源性比对分析,在嗜热藻（Thermosynechococcus elongatus BP-1）里面找到了与已知的Pb/Pg型蓝细菌光敏色素TePixJ和TeTlr0924同源的3个基因tlr0911、tlr1215和tlr1999。通过分子克隆技术把它们的GAF结构域分别构建在pET30a（＋）表达载体上,与可生成藻蓝胆素（PCB）的质粒pACYCDuet-ho1-pcyA在大肠杆菌BL21（DE3）体内重组,生成重组蛋白,利用亲和层析柱分离纯化,纯化后的蛋白质经过锌荧光和蛋白质酸性尿素变性以及荧光光谱和吸收光谱等实验分析鉴定,结果表明,Tlr0911-GAF存在蓝光吸收态Pb406 nm和绿光吸收态Pg527 nm之间的可逆光转换,它可共价结合两种藻胆色素,即藻紫胆素（PVB）和藻蓝胆素（PCB）,Tlr1999-GAF则存在蓝光吸收态Pb417 nm和青光吸收态Pt496 nm之间的可逆光转换,它同样共价结合PVB和PCB,而Tlr1215-GAF1和Tlr1215-GAF2不能自发结合藻胆色素,不具有光活性。     

The cyanobacteriochrome, TePixJ, isomerizes its own chromophore by converting phycocyanobilin to phycoviolobilin

The cyanobacterial phototaxis regulator protein, TePixJ, is a member of the subfamily of cyanobacteriochromes that binds phycoviolobilin (PVB) as a chromophore and exhibits reversible photoconversion between blue light-absorbing (Pb) and green light-absorbing (Pg) forms. We reconstituted the PVB-binding photoactive holocomplex in vivo and in vitro. Coexpression of the apoprotein and phycocyanobilin (PCB) in Escherichia coli (in vivo reconstitution) produced a mixture of the PCB-bound and PVB-bound holoproteins. Reconstitution in vitro of the apoprotein and synthetic PCB quickly generated a photoactive complex, which covalently bound PCB and exhibited partially reversible photoconversion between two species by UV-vis spectroscopy (with a λ(max) values of 430 and 545 nm). Further incubation produced slow isomerization of PCB to PVB with concomitant improvement of photoreactivity. Site-directed mutagenesis confirmed that Cys522, and a second conserved Cys (Cys494), are both essential for the assembly of the photoactive complex. Fourier transform infrared (FTIR) spectroscopy revealed green light-induced cross-linking, and blue light-induced release, of a thiol group, possibly that of Cys494. These results suggest that the Pb/Pg-type cyanobacteriochrome TePixJ is assembled in at least three steps: (i) rapid and stable chromophorylation of PCB, (ii) additional photoreversible chromophorylation, and (iii) subsequent slow isomerization of PCB to PVB. In addition to its known autolyase activity with Cys522 and photoreversible isomerase activity (of the Z and E isomers at C15 and C16 of PCB), the GAF domain of TePixJ therefore appears to have other roles: as an isomerase (converting PCB to PVB) and as a photoreversible autolyase with a second conserved Cys residue.     

Characterization of cyanobacteriochrome TePixJ from a thermophilic cyanobacterium Thermosynechococcus elongatus strain BP-1

A putative photoreceptor gene, TepixJ, of a thermophilic cyanobacterium is homologous to SypixJ1 that mediates positive phototaxis in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803. The putative chromophore-binding GAF domain of TePixJ protein was overexpressed as a fusion with a polyhistidine tag (His-TePixJ_GAF) in Synechocystis cells and isolated to homogeneity. The photoreversible conversion of His-TePixJ_GAF showed peaks at 531, 341 and 266 nm for the green light-absorbing form (Pg form), and peaks at 433 and 287 nm for the blue light-absorbing form (Pb form). At 77K, the Pg form fluoresced at 580 nm, while the Pb form did not emit any fluorescence. Mass spectrometry of the tryptic chromopeptide demonstrated that a phycocyanobilin isomer binds to the conserved cysteine at ring A via a thioether bond. It is established that TePixJ and SyPixJ1 are novel photoreceptors in cyanobacteria ('cyanobacteriochromes') that are similar, but distinct from the phytochromes and bacteriophytochromes.     

Characterization of two thermostable cyanobacterial phytochromes reveals global movements in the chromophore-binding domain during photoconversion

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

Mutational analysis of Deinococcus radiodurans bacteriophytochrome reveals key amino acids necessary for the photochromicity and proton exchange cycle of phytochromes

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

Cyanochromes Are Blue/Green Light Photoreversible Photoreceptors Defined by a Stable Double Cysteine Linkage to a Phycoviolobilin-type Chromophore

Phytochromes are a collection of bilin-containing photoreceptors that regulate a diverse array of processes in microorganisms and plants through photoconversion between two stable states, a red light-absorbing Pr form, and a far red light-absorbing Pfr form. Recently, a novel set of phytochrome-like chromoproteins was discovered in cyanobacteria, designated here as cyanochromes, that instead photoconvert between stable blue and green light-absorbing forms Pb and Pg, respectively. Here, we show that the distinctive absorption properties of cyanochromes are facilitated through the binding of phycocyanobilin via two stable cysteine-based thioether linkages within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. Absorption, resonance Raman and infrared spectroscopy, and molecular modeling of the Te-PixJ GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domain assembled with phycocyanobilin are consistent with attachments to the C3¹ carbon of the ethylidene side chain and the C4 or C5 carbons in the A–B methine bridge to generate a double thioether-linked phycoviolobilin-type chromophore. These spectroscopic methods combined with NMR data show that the bilin is fully protonated in the Pb and Pg states and that numerous conformation changes occur during Pb → Pg photoconversion. Also identified were a number of photochromically inactive mutants with strong yellow or red fluorescence that may be useful for fluorescence-based cell biological assays. Phylogenetic analyses detected cyanochromes capable of different signaling outputs in a wide range of cyanobacterial species. One unusual case is the Synechocystis cyanochrome Etr1 that also binds ethylene, suggesting that it works as a hybrid receptor to simultaneously integrate light and hormone signals.Phytochromes (Phys)³ comprise a large and diverse superfamily of photoreceptors that regulate a wide range of physiological responses in plants, fungi, bacteria, and cyanobacteria (1–3). They are unique among photoreceptors by being able to photoconvert between two stable states, a red light-absorbing Pr form that is typically the dark-adapted and biologically inactive conformer and a far-red light-absorbing Pfr form that requires light for its production and is typically the biologically active conformer. By interconverting between Pr and Pfr, Phys act as light-regulated switches in controlling processes ranging from phototaxis and pigmentation in bacteria to seed germination, photomorphogenesis, and flowering time in higher plants.Light absorption by Phys is directed by a bilin (or linear tetrapyrrole) chromophore produced by the oxidative cleavage of heme. Although bacterial and fungal Phys use the immediate cleavage product biliverdin (BV), cyanobacterial and higher plant Phys use phycocyanobilin (PCB) and phytochromobilin, respectively, produced by enzymatic reduction of BV (1, 2). The bilin is then covalently bound autocatalytically to the photosensory unit of the apoprotein, which typically contains a sequence of Per/Arndt/Sim (PAS), cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF), and Phy-associated (PHY) domains. Intimate contact between the bilin and surrounding protein residues then generates the unique photochromic properties of Phys. Recent three-dimensional structures of the Pr form of several bacterial Phys (BphPs) and two cyanobacterial Phys (Cphs) have shown that the bilin is deeply buried within the GAF domain in a ZZZssa configuration and that the connection between the GAF and PAS domains is stabilized by a rare figure-of-eight knot involving the region upstream of the PAS domain being lassoed by a conserved loop within the GAF domain (4–9). Although the structure of Pfr remains unsolved, various physicochemical studies have proposed that photoconversion involves a rotation of one of the three methine bridges between the pyrrole rings (1, 10–14). This rotation then induces much slower thermally driven movements of the protein to initiate signal transduction.In microorganisms, Pfr can activate a variety of signaling systems using output motifs directly appended to the C-terminal end of the photosensory region. The most prevalent are histidine kinase domains that then begin specific two-component phosphorelays (3, 15, 16). Although the output of plant Phys remains unclear, the presence of a C-terminal HK-related domain suggests that they also work as light-regulated protein kinases (17).In addition to the canonical Phys, it has become apparent through phylogenetic and biochemical studies that a heterogeneous collection of Phy-like photoreceptors exists (e.g. Refs. 3 and 18). These include Phys that prefer Pfr as the dark-adapted state (7, 19, 20), Phys that photoconvert from Pr to shorter wavelength-absorbing “near red” or Pnr forms (6, 21), and Phy-like photoreceptors that bind bilins but instead photoconvert between forms with maximal absorption other than red and far-red light (22–25). Often these Phy-like sequences are missing key residues or domains common among canonical Phys, suggesting that they employ novel bilins as chromophores, bind the bilin in different architectures, and/or use distinct photochemistries.One subclass of novel Phy-like photoreceptors present in a number of cyanobacteria, which we have designated cyanochromes (or Cycs) to better distinguish them from Cphs, is exemplified by Synechocystis sp. PCC6803 (Syn) PixJ (or TaxD1, locus sll0041) and its relatives. Syn-PixJ was discovered based on its involvement in blue light-mediated phototaxis in this mesophilic cyanobacterium (26, 27) with its close homolog Te-PixJ (locus tll0569) then found in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 by sequence similarity (28). Like Cphs, the cyanochromes tested thus far covalently bind PCB but then generate photoreceptors that convert between blue and green light-absorbing forms designated Pb and Pg, respectively (22, 24, 29). Subsequent studies proposed that PCB is converted to phycoviolobilin (PVB) upon attachment to the apoprotein (30). PVB differs from PCB by having a methylene instead of a methine bridge between the A and B pyrrole rings, which blue-shifts the absorption of the chromophore by shortening the π-conjugation system. Phototransformation of Pb to Pg could then occur by a mechanism similar to Phys.How Te-PixJ and related cyanochromes bind PCB to generate more blue-shifted PVB-type chromophores remains unclear. Like Cphs, two cyanochromes examples link PCB via a thioether linkage between a cysteine in the Cyc-GAF domain and the C3¹ carbon of the ethylidene side chain of ring A (24, 28). Additionally, loss of the C4C5 double bond is necessary to generate PVB. One model by Ishizuka et al. (30) from studies with Te-PixJ proposed that the double bond moves from the C4-C5 position to the C2-C3 position by an autoisomerase activity intrinsic to the GAF domain. A more recent model by Rockwell et al. (24) using another Syn-PixJ relative in T. elongatus, Tlr0924, invoked the possibility of a second cysteine that also participates in PCB ligation. This cysteine was proposed to bind the bilin at the C10 position via a reversible thioether linkage. In the dark-adapted Pb state, the second linkage would then be formed to generate a rubin-like chromophore attached to the bridge between the B and C pyrrole rings. This bond would then break upon photoconversion to generate the more π-conjugated green light-absorbing photoproduct Pg.In this report, we employed a number of physicochemical approaches to help resolve the unique chromophore architecture and photochemical properties of cyanochromes, using Te-PixJ as the example. By independently mutagenizing the cysteine that binds the A ring ethylidene (Cys-522 (22)) and that proposed by Rockwell et al. (24) to reversibly bind the bilin at a second site (Cys-494), we demonstrate that both residues form light-stable covalent adducts with a PVB-type chromophore. In addition, we employed various spectroscopic methods to show that the bound PVB is fully protonated as both Pb and Pg, that only one pyrrole ring is active during photoconversion, and that the polypeptide may undergo extensive remodeling as Pb converts to Pg. We identified a set of conserved amino acids in Te-PixJ important for cyanochrome photochemistry, including several that when substituted generate yellow or red fluorescent chromoproteins potentially useful for cell biological applications. Phylogenetic analyses show that cyanochromes are widespread among cyanobacteria with their closest relatives being members of the red/far-red light-absorbing Phy subfamily defined by the absence of the N-terminal PAS domain (31).     

Solution structure of a cyanobacterial phytochrome GAF domain in the red-light-absorbing ground state

The unique photochromic absorption behavior of phytochromes (Phys) depends on numerous reversible interactions between the bilin chromophore and the associated polypeptide. To help define these dynamic interactions, we determined by NMR spectroscopy the first solution structure of the chromophore-binding cGMP phosphodiesterase/adenylcyclase/FhlA (GAF) domain from a cyanobacterial Phy assembled with phycocyanobilin (PCB). The three-dimensional NMR structure of Synechococcus OS-B′ cyanobacterial Phy 1 in the red-light-absorbing state of Phy (Pr) revealed that PCB is bound to Cys138 of the GAF domain via the A-ring ethylidene side chain and is buried within the GAF domain in a ZZZsyn,syn,anti configuration. The D ring of the chromophore sits within a hydrophobic pocket and is tilted by approximately 80° relative to the B/C rings by contacts with Lys52 and His169. The solution structure revealed remarkable flexibility for PCB and several adjacent amino acids, indicating that the Pr chromophore has more freedom in the binding pocket than anticipated. The propionic acid side chains of rings B and C and Arg101 and Arg133 nearby are especially mobile and can assume several distinct and energetically favorable conformations. Mutagenic studies on these arginines, which are conserved within the Phy superfamily, revealed that they have opposing roles, with Arg101 and Arg133 helping stabilize and destabilize the far-red-light-absorbing state of Phy (Pfr), respectively. Given the fact that the Synechococcus OS-B′ GAF domain can, by itself, complete the Pr → Pfr photocycle, it should now be possible to determine the solution structure of the Pfr chromophore and surrounding pocket using this Pr structure as a framework.     

Unusual Spectral Properties of Bacteriophytochrome Agp2 Result from a Deprotonation of the Chromophore in the Red-absorbing Form Pr

Phytochromes are widely distributed photoreceptors with a bilin chromophore that undergo a typical reversible photoconversion between the two spectrally different forms, Pr and Pfr. The phytochrome Agp2 from Agrobacterium tumefaciens belongs to the group of bathy phytochromes that have a Pfr ground state as a result of the Pr to Pfr dark conversion. Agp2 has untypical spectral properties in the Pr form reminiscent of a deprotonated chromophore as confirmed by resonance Raman spectroscopy. UV/visible absorption spectroscopy showed that the pK_a is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pK_a shift of more than 3 units. The Pr/Pfr ratio after saturating irradiation with monochromatic light is strongly pH-dependent. This is partially due to a back-reaction of the deprotonated Pr chromophore at pH 9 after photoexcitation as found by flash photolysis. The chromophore protonation and dark conversion were affected by domain swapping and site-directed mutagenesis. A replacement of the PAS or GAF domain by the respective domain of the prototypical phytochrome Agp1 resulted in a protonated Pr chromophore; the GAF domain replacement afforded an inversion of the dark conversion. A reversion was also obtained with the triple mutant N12S/Q190L/H248Q, whereas each single point mutant is characterized by decelerated Pr to Pfr dark conversion.     

蓝藻红色荧光蛋白All1280 GAF2在E.coli BL21(DE3)中的表达及其突变体构建

采用PCR技术从鱼腥藻（Anabaena sp.）PCC 7120中扩增获得红色荧光蛋白基因all1280 gaf2,并利用BamHⅠ和SalⅠ酶切位点,将该基因插入到pET-30a（+）中,构建表达载体pET-all1280 gaf2。将该表达载体与藻胆色素生物合成质粒pACYC-ho1-pcyA同时转化到大肠杆菌E.coli BL21（DE3）,表达后获得大肠杆菌色素细胞。结果显示,该色素细胞在荧光显微镜下具有红色荧光,且在15E/15Z态之间具有可逆光效应。进一步以pET-all1280 gaf2为模板,通过定点突变技术在all1280 gaf2基因中引入C53A突变,获得了突变体All1280 GAF2（C53A）。将All1280 GAF2（C53A）与藻胆色素在E.coli BL21（DE3）中共表达,获得了比野生型红色荧光更强的大肠杆菌色素细胞。研究结果表明,与野生型相比,All1280 GAF2（C53A）具有较高的摩尔消光系数和荧光量子产率,红色荧光更强。     

蓝藻红色荧光蛋白All1280 GAF2在E. coli BL21(DE3)中的表达及其突变体构建（英文）

采用PCR技术从鱼腥藻(Anabaena sp.) PCC 7120中扩增获得红色荧光蛋白基因all1280 gaf2,并利用Bam HⅠ和SalⅠ酶切位点,将该基因插入到pET-30a(+)中,构建表达载体pET-all1280 gaf2。将该表达载体与藻胆色素生物合成质粒pACYC-ho1-pcyA同时转化到大肠杆菌E. coli BL21 (DE3),表达后获得大肠杆菌色素细胞。结果显示,该色素细胞在荧光显微镜下具有红色荧光,且在15E/15Z态之间具有可逆光效应。进一步以pET-all1280 gaf2为模板,通过定点突变技术在all1280 gaf2基因中引入C53A突变,获得了突变体All1280 GAF2 (C53A)。将All1280 GAF2 (C53A)与藻胆色素在E. coli BL21 (DE3)中共表达,获得了比野生型红色荧光更强的大肠杆菌色素细胞。研究结果表明,与野生型相比,All1280 GAF2 (C53A)具有较高的摩尔消光系数和荧光量子产率,红色荧光更强。     

Crystallographic and Electron Microscopic Analyses of a Bacterial Phytochrome Reveal Local and Global Rearrangements during Photoconversion

Phytochromes are multidomain photoswitches that drive light perception in plants and microorganisms by coupling photoreversible isomerization of their bilin chromophore to various signaling cascades. How changes in bilin conformation affect output by these photoreceptors remains poorly resolved and might include several species-specific routes. Here, we present detailed three-dimensional models of the photosensing module and a picture of an entire dimeric photoreceptor through structural analysis of the Deinococcus radiodurans phytochrome BphP assembled with biliverdin (BV). A 1.16-Å resolution crystal structure of the bilin-binding pocket in the dark-adapted red light-absorbing state illuminated the intricate network of bilin/protein/water interactions and confirmed the protonation and ZZZssa conformation of BV. Structural and spectroscopic comparisons with the photochemically compromised D207A mutant revealed that substitutions of Asp-207 allow inclusion of cyclic porphyrins in addition to BV. A crystal structure of the entire photosensing module showed a head-to-head, twisted dimeric arrangement with bowed helical spines and a hairpin protrusion connecting the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) and phytochrome-specific (PHY) domains. A key conserved hairpin feature is its anti-parallel, two β-strand stem, which we show by mutagenesis to be critical for BphP photochemistry. Comparisons of single particle electron microscopic images of the full-length BphP dimer in the red light-absorbing state and the photoactivated far-red light-absorbing state revealed a large scale reorientation of the PHY domain relative to the GAF domain, which alters the position of the downstream histidine kinase output module. Together, our data support a toggle model whereby bilin photoisomerization alters GAF/PHY domain interactions through conformational modification of the hairpin, which regulates signaling by impacting the relationship between sister output modules.     

Photophysical diversity of two novel cyanobacteriochromes with phycocyanobilin chromophores: photochemistry and dark reversion kinetics

Cyanobacteriochromes are phytochrome homologues in cyanobacteria that act as sensory photoreceptors. We compare two cyanobacteriochromes, RGS (coded by slr1393) from Synechocystis sp. PCC 6803 and AphC (coded by all2699) from Nostoc sp. PCC 7120. Both contain three GAF (cGMP phosphodiesterase, adenylyl cyclase and FhlA protein) domains (GAF1, GAF2 and GAF3). The respective full-length, truncated and cysteine point-mutated genes were expressed in Escherichia coli together with genes for chromophore biosynthesis. The resulting chromoproteins were analyzed by UV-visible absorption, fluorescence and circular dichroism spectroscopy as well as by mass spectrometry. RGS shows a red-green photochromism (λ(max) = 650 and 535 nm) that is assigned to the reversible 15Z/E isomerization of a single phycocyanobilin-chromophore (PCB) binding to Cys528 of GAF3. Of the three GAF domains, only GAF3 binds a chromophore and the binding is autocatalytic. RGS autophosphorylates in vitro; this reaction is photoregulated: the 535 nm state containing E-PCB was more active than the 650 nm state containing Z-PCB. AphC from Nostoc could be chromophorylated at two GAF domains, namely GAF1 and GAF3. PCB-GAF1 is photochromic, with the proposed 15E state (λ(max) = 685 nm) reverting slowly thermally to the thermostable 15Z state (λ(max) = 635 nm). PCB-GAF3 showed a novel red-orange photochromism; the unstable state (putative 15E, λ(max) = 595 nm) reverts very rapidly (τ ~ 20 s) back to the thermostable Z state (λ(max) = 645 nm). The photochemistry of doubly chromophorylated AphC is accordingly complex, as is the autophosphorylation: E-GAF1/E-GAF3 shows the highest rate of autophosphorylation activity, while E-GAF1/Z-GAF3 has intermediate activity, and Z-GAF1/Z-GAF3 is the least active state.     

Phototransformation of the Red Light Sensor Cyanobacterial Phytochrome 2 from Synechocystis Species Depends on Its Tongue Motifs

Phytochromes are photoreceptors using a bilin tetrapyrrole as chromophore, which switch in canonical phytochromes between red (P_r) and far red (P_fr) light-absorbing states. Cph2 from Synechocystis sp., a noncanonical phytochrome, harbors besides a cyanobacteriochrome domain a second photosensory module, a P_r/P_fr-interconverting GAF-GAF bidomain (SynCph2(1-2)). As in the canonical phytochromes, a unique motif of the second GAF domain, the tongue region, seals the bilin-binding site in the GAF1 domain from solvent access. Time-resolved spectroscopy of the SynCph2(1-2) module shows four intermediates during P_r → P_fr phototransformation and three intermediates during P_fr → P_r back-conversion. A mutation in the tongue''s conserved PRXSF motif, S385A, affects the formation of late intermediate R3 and of a P_fr-like state but not the back-conversion to P_r via a lumi-F-like state. In contrast, a mutation in the likewise conserved WXE motif, W389A, changes the photocycle at intermediate R2 and causes an alternative red light-adapted state. Here, back-conversion to P_r proceeds via intermediates differing from SynCph2(1-2). Replacement of this tryptophan that is ∼15 Å distant from the chromophore by another aromatic amino acid, W389F, restores native P_r → P_fr phototransformation. These results indicate large scale conformational changes within the tongue region of GAF2 during the final processes of phototransformation. We propose that in early intermediates only the chromophore and its nearest surroundings are altered, whereas late changes during R2 formation depend on the distant WXE motifs of the tongue region. Ser-385 within the PRXSF motif affects only late intermediate R3, when refolding of the tongue and docking to the GAF1 domain are almost completed.     

Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803. Purification, assembly, and quaternary structure.

The phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 forms holoprotein adducts with close spectral similarity to plant phytochromes when autoassembled in vitro with bilin chromophores. Cph1 is a 85-kDa protein that acts as a light-regulated histidine kinase seemingly involved in 'two-component' signalling. This paper describes the improvement of Cph1 purification, estimation of the extinction coefficient of holo-Cph1, spectral analyses of the assembly procedure and studies on quaternary structure. During assembly with the natural chromophore phycocyanobilin (PCB), a red-shifted intermediate is observed. A similar result was obtained when phycoerythrobilin was used as chromophore. As shown by SDS/PAGE and Zn2+ fluorescence, the covalent attachment of PCB is blocked by 1 mM iodoacetamide, a cysteine-derivatizing agent. When PCB was incubated with blocked apo-Cph1, again a shoulder at longer wavelengths appeared. It is therefore proposed that the long-wavelength-absorbing form represents the protonated, noncovalently bound bilin. Biliverdin, which is neither protonated nor covalently attached, undergoes spectral changes in its blue-absorbing band upon incubation with apo-Cph1. On the basis of these data we therefore propose a three-step model for phytochrome autoassembly. Size-exclusion chromatography revealed different mobilities for the apoprotein, red-absorbing Cph1-PCB and far-red-absorbing Cph1-PCB. The major peaks of both holoprotein adducts had apparent molecular masses approximately 200 kDa, a result in agreement with the notion that autophosphorylation in sensory histidine kinases requires dimerization. When Cph1-PCB was further purified by preparative native electrophoresis, the mobility on size-exclusion chromatography was approximately 100 kDa, and it was found to have lost its kinase activity, results implying that the material had lost its capacity to dimerize.     

蓝细菌光敏色素Alr1966GAF2及其突变体的表达与光化学性质研究

采用PCR技术从鱼腥藻（Anabaena sp.PCC7120）中扩增蓝细菌光敏色素基因片段alr1966gaf2,将alr1966gaf2插入到pET-30a（+）载体中,构建表达质粒pET-alr1966gaf2。最后将Alr1966GAF2与HO1、PcyA在E.coli BL21（DE3）中共表达获得色素蛋白Alr1966GAF2,并对该蛋白的光化学性质进行分析。结果显示,色素蛋白Alr1966GAF2结合色素为藻蓝胆素（phycoerythrobilin,PCB）或藻紫胆素（phycoviolobilin,PVB）,在3种不同吸收态15Z-P_{428 nm}、中间态和15E-P_{514 nm}之间具有顺序可逆光效应。通过定点突变技术将DXCF基序中的保守性Cys突变为Ala,获得了突变体Alr1966GAF2（C72A）。将Alr1966GAF2（C72A）与HO1、PcyA共表达,获得色素蛋白Alr1966GAF2（C72A）。研究结果表明Alr1966GAF2（C72A）结合色素为PCB,Alr1966GAF2（C72A）-PCB具有较强的荧光活性,其荧光量子的产率高达0.11。Alr1966GAF2（C72A）不仅能够共价结合PCB,还可以结合胆绿素（Biliverdin,BV）,均具有较强的红色荧光活性。     

Fluorescence of phytochrome adducts with synthetic locked chromophores

We performed steady state fluorescence measurements with phytochromes Agp1 and Agp2 of Agrobacterium tumefaciens and three mutants in which photoconversion is inhibited. These proteins were assembled with the natural chromophore biliverdin (BV), with phycoerythrobilin (PEB), which lacks a double bond in the ring C-D-connecting methine bridge, and with synthetic bilin derivatives in which the ring C-D-connecting methine bridge is locked. All PEB and locked chromophore adducts are photoinactive. According to fluorescence quantum yields, the adducts may be divided into four different groups: wild type BV adducts exhibiting a weak fluorescence, mutant BV adducts with about 10-fold enhanced fluorescence, adducts with locked chromophores in which the fluorescence quantum yields are around 0.02, and PEB adducts with a high quantum yield of around 0.5. Thus, the strong fluorescence of the PEB adducts is not reached by the locked chromophore adducts, although the photoconversion energy dissipation pathway is blocked. We therefore suggest that ring D of the bilin chromophore, which contributes to the extended π-electron system of the locked chromophores, provides an energy dissipation pathway that is independent on photoconversion.