Assembly of synthetic locked chromophores with agrobacterium phytochromes Agp1 and Agp2

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red-absorbing form Pr and the far-red-absorbing form Pfr. Agrobacterium tumefaciens has two phytochromes, Agp1 and Agp2, with antagonistic properties: in darkness, Agp1 converts slowly from Pfr to Pr, whereas Agp2 converts slowly from Pr to Pfr. In a previous study, we have assembled Agp1 with synthetic locked chromophores 15Za, 15Zs, 15Ea, and 15Es in which the C15=C16 double bond is fixed in either the E or Z configuration and the C14-C15 single bond is fixed in either the syn (s) or anti (a) conformation. In the present study, the locked chromophores 5Za and 5Zs were used for assembly with Agp1; in these chromophores, the C4=C5 double bond is fixed in the Z configuration, and the C5-C6 single bond is fixed in either the syn or anti conformation. All locked chromophores were also assembled with Agp2. The data showed that in both phytochromes the Pr chromophore adopts a C4=C5 Z C5-C6 syn C15=C16 Z C14-C15 anti stereochemistry and that in the Pfr chromophore the C15=C16 double bond has isomerized to the E configuration, whereas the C14-C15 single bond remains in the anti conformation. Photoconversion shifted the absorption maxima of the 5Zs adducts to shorter wavelengths, whereas the 5Za adducts were shifted to longer wavelengths. Thus, the C5-C6 single bond of the Pfr chromophore is rather in an anti conformation, supporting the previous suggestion that during photoconversion of phytochromes, a rotation around the ring A-B connecting single bond occurs.     

Sterically locked synthetic bilin derivatives and phytochrome Agp1 from Agrobacterium tumefaciens form photoinsensitive Pr- and Pfr-like adducts

Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15=C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15=C16 double bond is in either the Z or E configuration and the C14-C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15=C16 Z configuration and a C14-C15 anti conformation in the Pr form and a C15=C16 E configuration and a C14-C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.     

Locked 5Zs-biliverdin blocks the Meta-RA to Meta-RC transition in the functional cycle of bacteriophytochrome Agp1

The bacteriophytochrome Agp1 was reconstituted with a locked 5Zs-biliverdin in which the C(4)=C(5) and C(5)-C(6) bonds of the methine bridge between rings A and B are fixed in the Z and syn configuration/conformation, respectively. In Agp1-5Zs the photoconversion proceeds via the Lumi-R intermediate to Meta-R(A), but the following millisecond-transition to Meta-R(C) is blocked. Consistently, no transient proton release was detected. The photoconversion of Agp1-5Zs is apparently arrested in a Meta-R(A)-like intermediate, since the subsequent syn to anti rotation around the C(5)-C(6) bond is prevented by the lock. The Meta-R(A)-like photoproduct was characterized by its distinctive CD spectrum suggesting a reorientation of ring D.     

Agrobacterium phytochrome as an enzyme for the production of ZZE bilins

Photoconversion of phytochrome from the red-absorbing form Pr to the far-red-absorbing form Pfr is initiated by a Z to E isomerization around the ring C-ring D connecting double bond; the chromophore undergoes a ZZZ to ZZE isomerization. In vivo, phytochrome chromophores are covalently bound to the protein, but several examples of noncovalent in vitro adducts have been reported which also undergo Pr to Pfr photoconversion. We show that free biliverdin or phycocyanobilin, highly enriched in the ZZE isomer, can easily be obtained from chromophores bound in a noncovalent manner to Agrobacterium phytochrome Agp1, and used for spectral assays. Photoconversion of free biliverdin in a methanol/HCl solution from ZZE to ZZZ proceeded with a quantum yield of 1.8%, but was negligible in neutral methanol solution, indicating that this process is proton-dependent. The ZZE form of biliverdin and phycocyanobilin were tested for their ability to assemble with Agp1 and cyanobacterial phytochrome Cph1, respectively. In both cases, a Pfr-like adduct was formed but the chromophore was bound in a noncovalent manner to the protein. Agp1 Pfr undergoes dark reversion to Pr; the same feature was found for the noncovalent ZZE adduct. After dark reversion, the chromophore became covalently bound to the protein. In analogy, the PCB chromophore became covalently bound to Cph1 upon irradiation with strong far-red light which initiated ZZE to ZZZ isomerization. Agrobacterium Agp2 belongs to a yet small group of phytochromes which also assemble in the Pr form but convert from Pr to Pfr in darkness. When the Agp2 apoprotein was assembled with the ZZE form of biliverdin, the formation of the final adduct was accelerated compared to the formation of the ZZZ control, indicating that the ZZE chromophore fits directly into the chromophore pocket of Agp2.     

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.     

A Polarity Probe for Monitoring Light-induced Structural Changes at the Entrance of the Chromophore Pocket in a Bacterial Phytochrome

Light-induced structural changes at the entrance of the chromophore pocket of Agp1 phytochrome were investigated by using a thiol-reactive fluorescein derivative that is covalently attached to the genuine chromophore binding site (Cys-20) and serves as a polarity probe. In the apoprotein, the absorption spectrum of bound fluorescein is red-shifted with respect to that of the free label suggesting that the probe enters the hydrophobic chromophore pocket. Assembly of this construct with the chromophores phycocyanobilin or biliverdin is associated with a blue-shift of the fluorescein absorption band indicating the displacement of the probe out of the pocket. The probe does not affect the photochromic and kinetic properties of the noncovalent bilin adducts. Upon photoconversion to P_fr, the probe spectrum undergoes again a bathochromic shift and a strong rise in CD indicating a more hydrophobic and asymmetric environment. We propose that the environmental changes of the probe reflect conformational changes at the entrance of the chromophore pocket and are indicative for rearrangements of the chromophore ring A. Flash photolysis measurements showed that the absorption changes of the probe are kinetically coupled to the formation of Meta-R_C and P_fr. In the biliverdin adduct, an additional component occurs that probably reflects a transition between two Meta-R_C substates. Analogous results to that of the noncovalent phycocyanobilin adduct were obtained with the mutant V249C in which probe and chromophore are covalently attached. The conformational changes of the chromophore are correlated to proton transfer to the protein surface.Phytochromes are red-light photoreceptors occurring in plants, bacteria, and fungi where they control important developmental processes (1–6). The discovery of microbial phytochromes from genome sequencing (7–9) provided new prospects for biochemical, spectroscopic and structural analyses of this light sensor family. Agp1 (AtBphP1)³ from the soil bacterium Agrobacterium tumefaciens is a typical member of the widespread family of proteobacterial phytochromes (10, 11) and is the subject of the present study.The domain arrangement of canonical phytochromes consists of an N-terminal photosensory domain, including PAS, GAF, and PHY domains and a C-terminal regulatory kinase domain (see, e.g. Ref. 3). Bacterial phytochromes lack the N-terminal extension, and the PAS module insertion of plant phytochromes (3). In most of the bacterial phytochromes, the C-terminal regulatory domain is a histidine kinase (4). These kinases form homodimers as functional units (12) where the subunits transphosphorylate each other (13). The cofactors are linear tetrapyrroles that are covalently attached via a thioether linkage (14) to the side chains of specific conserved cysteine residues. The native chromophore of plant phytochromes is phytochromobilin (PΦB) (14), some cyanobacterial phytochromes incorporate phycocyanobilin (PCB) (15, 16), and all other bacterial phytochromes bind biliverdin (BV) (10, 11). Whereas the chromophore binding site of the more reduced bilins PΦB and PCB is located in the GAF domain, the binding site of BV is close to the N terminus upstream of the PAS domain (4, 11). The two distinct binding sites apparently require a specific substituent at the C3 carbon of pyrrole ring A, either an ethylidene (PΦB and PCB) or a vinyl (BV) group, for covalent attachment of the bilin chromophore (4). The holophytochrome assembly that includes covalent attachment of the chromophore is an autocatalytic process implying an intrinsic bilin C-S lyase activity of the apophytochrome (17). Kinetic studies of the autoassembly in vitro showed that ligation of the chromophore is the ultimate step following incorporation in the binding pocket and internal protonation (18).Phytochromes display photochromicity involving two either thermally stable or long-lived states, P_r and P_fr (red and far-red absorbing forms), that can be reversibly converted by light of appropriate wavelengths. The P_r to P_fr photoconversion is initiated by a rapid Z/E isomerization of the C-D methine bridge of the bilin chromophore (19–22) leading within picoseconds to the formation of the Lumi-R intermediate (23, 24). The following thermal relaxations via Meta-R_A and Meta-R_C intermediates to P_fr proceed on the time scale of microseconds and milliseconds (25–28).Assembly of Agp1 with locked BV derivatives showed that the geometry of the C-D methine bridge is 15Z_anti in P_r and 15E_anti in P_fr (29) suggesting that this methine bridge remains in the anti conformation during photoconversion. The crystal structures of the chromophore binding domains of the bacteriophytochromes from Deinococcus radiodurans and Rhodopseudomonas palustris revealed that the BV chromophore adopts a 5Z_syn,10Z_syn,15Z_anti configuration/conformation in the P_r state (30–32). The 5Z_syn geometry of the A-B methine bridge in the P_r state was confirmed by assembly of Agp1 with the corresponding locked BV chromophore (33). Recently, heteronuclear NMR investigations and crystallographic studies on the complete photosensory domain of the cyanobacterial phytochrome Cph1 from Synechocystis showed that the PCB chromophore is also in the 5Z_syn,10Z_syn,15Z_anti geometry in P_r (34, 35).Because the locked 5Z_syn adduct of Agp1 did not show a P_fr-like photo-product, conformational changes of the A-B methine bridge in the thermal relaxation cascade have been predicted (33). Flash photolysis experiments with this adduct suggested that these changes occur in the Meta-R_A to Meta-R_C transition (36). The stereochemistry of the A-B methine bridge in the P_fr state and in the preceding intermediates could not be determined unambiguously yet. Recent studies with doubly locked chromophores suggest that the C5–C6 single bond undergoes a thermal rotation from syn to anti in the photoconversion of Agp1, whereas an additional Z/E isomerization around the C4C5 double bond (hula-twist mechanism) was postulated for Agp2 (37). However, the crystal structure of the photosensory domain of the bacteriophytochrome PaBphP in its P_fr-enriched dark-adapted state favors the 5Z_syn conformation of the BV chromophore (38). Structural changes of the A-B methine bridge were excluded for the PCB chromophore of Cph1 on the basis of heteronuclear NMR (34), whereas low temperature Fourier transform IR studies on plant phytochrome suggested an environmental change of the ring A carbonyl group and/or a twist of the A-B methine bridge (39).The mechanism by which the signal is transmitted from the bilin chromophore to the protein is still obscure. The recent three-dimensional structures of the complete photosensory domains of Cph1 (35) and PaBphP (38) reveal key interactions between GAF and PHY domains in the corresponding dark states reflecting P_r and P_fr, respectively. In view of the intrinsic differences between the two phytochromes, it is not trivial to differentiate which of the numerous structural differences arise from light-induced conformational changes and are thus potentially important for signal transmission. We note that many approaches to provide a clue on the mechanism of signal transmission from the bilin chromophore to its proximate environment imply that this process is exclusively coupled to the photo-isomerization localized at ring D and its environment and that the chromophore then remains a passive element in the thermal relaxation cascade. This point of view is supported by recent results from femtosecond stimulated Raman spectroscopy suggesting that the chromophore structures in Lumi-R and P_fr are very similar (24). On the other hand, size exclusion chromatography experiments demonstrated that the global conformational changes observed for the P_fr state of Agp1 WT are absent in constructs (locked 5Zs adduct and mutants D197A and H250A), where the formation of P_fr is inhibited but the primary photoreaction proceeds (33, 40). These results are difficult to explain in terms of an ultra-fast signal transmission from the chromophore to the surrounding residues in its pocket.Light-induced conformational changes at the surface of plant phytochrome were observed by using covalently attached labels that are sensitive to the polarity of the microenvironment (41, 42). Due to the accessibility of several binding sites (i.e. the sulfhydryl groups of cysteines) in these experiments, the labeling was unspecific preventing further assignment of the observed changes to particular regions of the protein. Time-resolved absorption measurements with a covalently attached fluorescein derivative showed that the changes occur in the Meta-R_C to P_fr transition (41). In the present work with Agp1 phytochrome, we take advantage of the highly reactive sulfhydryl group of Cys-20, the genuine binding site of the BV chromophore, to specifically attach a fluorescein derivative. We observed that this construct assembles with PCB and BV forming noncovalent photochromic adducts, spectrally and kinetically undisturbed by the fluorescein label. Upon photo-conversion, the absorption band of the label displays a bathochromic shift and increase in ellipticity suggesting that the label moves in a more hydrophobic and asymmetric environment in the P_fr state. The label thus serves as a polarity probe at the entrance of the binding pocket. We postulate that these polarity changes reflect conformational changes of the A-B methine of the bilin chromophore and/or the microenvironment of ring A at the entrance of the binding pocket. Time-resolved measurements reveal that the changes occur in the Meta-R_A to Meta-R_C and Meta-R_C to P_fr transitions. Analogous results were obtained with the V249C mutant of Agp1 in which both the fluorescein probe and the PCB chromophore are covalently attached.     

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.     

Electronic transitions and heterogeneity of the bacteriophytochrome Pr absorption band: An angle balanced polarization resolved femtosecond VIS pump–IR probe study

Photoisomerization of biliverdin (BV) chromophore triggers the photoresponse in native Agp1 bacteriophytochrome. We discuss heterogeneity in phytochrome Pr form to account for the shape of the absorption profile. We investigated different regions of the absorption profile by angle balanced polarization resolved femtosecond VIS pump–IR probe spectroscopy. We studied the Pr form of Agp1 with its natural chromophore and with a sterically locked 18Et-BV (locked Agp1). We followed the dynamics and orientations of the carbonyl stretching vibrations of ring D and ring A in their ground and electronically excited states. Photoisomerization of ring D is reflected by strong signals of the ring D carbonyl vibration. In contrast, orientational data on ring A show no rotation of ring A upon photoexcitation. Orientational data allow excluding a ZZZasa geometry and corroborates a nontwisted ZZZssa geometry of the chromophore. We found no proof for heterogeneity but identified a new, to our knowledge, electronic transition in the absorption profile at 644 nm (S₀→S₂). Excitation of the S₀→S₂ transition will introduce a more complex photodynamics compared with S₀→S₁ transition. Our approach provides fundamental information on disentanglement of absorption profiles, identification of chromophore structures, and determination of molecular groups involved in the photoisomerization process of photoreceptors.     

Electronic transitions and heterogeneity of the bacteriophytochrome Pr?absorption band: An angle balanced polarization resolved femtosecond VIS pump–IR probe study

Photoisomerization of biliverdin (BV) chromophore triggers the photoresponse in native Agp1 bacteriophytochrome. We discuss heterogeneity in phytochrome Pr form to account for the shape of the absorption profile. We investigated different regions of the absorption profile by angle balanced polarization resolved femtosecond VIS pump–IR probe spectroscopy. We studied the Pr form of Agp1 with its natural chromophore and with a sterically locked 18Et-BV (locked Agp1). We followed the dynamics and orientations of the carbonyl stretching vibrations of ring D and ring A in their ground and electronically excited states. Photoisomerization of ring D is reflected by strong signals of the ring D carbonyl vibration. In contrast, orientational data on ring A show no rotation of ring A upon photoexcitation. Orientational data allow excluding a ZZZasa geometry and corroborates a nontwisted ZZZssa geometry of the chromophore. We found no proof for heterogeneity but identified a new, to our knowledge, electronic transition in the absorption profile at 644 nm (S₀→S₂). Excitation of the S₀→S₂ transition will introduce a more complex photodynamics compared with S₀→S₁ transition. Our approach provides fundamental information on disentanglement of absorption profiles, identification of chromophore structures, and determination of molecular groups involved in the photoisomerization process of photoreceptors.     

Crystallization and preliminary X-ray crystallographic analysis of the N-terminal photosensory module of phytochrome Agp1, a biliverdin-binding photoreceptor from Agrobacterium tumefaciens

Phytochromes are photochromic photoreceptors with a bilin chromophore that have been found in plants and bacteria. Typical bacterial phytochromes are composed of an N-terminal photosensory chromophore module and a C-terminal protein kinase. The former contains the chromophore, which allows phytochromes to adopt the two interconvertible spectral forms, Pr and Pfr. The N-terminal photosensory module of Agrobacterium phytochrome Agp1, Agp1-M15, was used for crystallization studies. The protein was either assembled with the natural chromophore biliverdin or a sterically locked synthetic biliverdin-derivative, termed 15Za. The last-named adduct does not undergo photoisomerization due to an additional carbon chain between the rings C and D of the chromophore. Both adducts could be crystallized, but the resolution was largely improved by the use of 15Za. Crystals of biliverdin-Agp1-M15 diffract to 6A resolution and belong to the tetragonal space group I422 with unit cell dimensions a = b = 171 Angstroms, c = 81 Angstroms, crystals of 15Za-Agp1-M15 belong to the same space group with similar unit cell dimensions a = b = 174 Angstroms, c = 80 Angstroms, but diffract to 3.4 Angstroms resolution. Assuming the asymmetric unit to be occupied by one monomer of 55kDa, the unit cell contains 54-55% solvent with a crystal volume per protein mass, V(m), of 2.7 Angstroms(3) Da(-1).     

Biliverdin binds covalently to agrobacterium phytochrome Agp1 via its ring A vinyl side chain

The widely distributed phytochrome photoreceptors carry a bilin chromophore, which is covalently attached to the protein during a lyase reaction. In plant phytochromes, the natural chromophore is coupled by a thioether bond between its ring A ethylidene side chain and a conserved cysteine residue within the so-called GAF domain of the protein. Many bacterial phytochromes carry biliverdin as natural chromophore, which is coupled in a different manner to the protein. In phytochrome Agp1 of Agrobacterium tumefaciens, biliverdin is covalently attached to a cysteine residue close to the N terminus (position 20). By testing different natural and synthetic biliverdin derivatives, it was found that the ring A vinyl side chain is used for chromophore attachment. Only those bilins that have ring A vinyl side chain were covalently attached, whereas bilins with an ethylidene or ethyl side chain were bound in a noncovalent manner. Phycocyanobilin, which belongs to the latter group, was however covalently attached to a mutant in which a cysteine was introduced into the GAF domain of Agp1 (position 249). It is proposed that the regions around positions 20 and 249 are in close contact and contribute both to the chromophore pocket. In competition experiments it was found that phycocyanobilin and biliverdin bind with similar strength to the wild type protein. However, in the V249C mutant, phycocyanobilin bound much more strongly than biliverdin. This finding could explain why during phytochrome evolution in cyanobacteria, the chromophore-binding site swapped from the N terminus into the GAF domain.     

Contrasting the excited-state dynamics of the photoactive yellow protein chromophore: protein versus solvent environments

Wavelength- and time-resolved fluorescence experiments have been performed on the photoactive yellow protein, the E46Q mutant, the hybrids of these proteins containing a nonisomerizing “locked” chromophore, and the native and locked chromophores in aqueous solution. The ultrafast dynamics of these six systems is compared and spectral signatures of isomerization and solvation are discussed. We find that the ultrafast red-shifting of fluorescence is associated mostly with solvation dynamics, whereas isomerization manifests itself as quenching of fluorescence. The observed multiexponential quenching of the protein samples differs from the single-exponential lifetimes of the chromophores in solution. The locked chromophore in the protein environment decays faster than in solution. This is due to additional channels of excited-state energy dissipation via the covalent and hydrogen bonds with the protein environment. The observed large dispersion of quenching timescales observed in the protein samples that contain the native pigment favors both an inhomogeneous model and an excited-state barrier for isomerization.     

Reconstitution of blue-green reversible photoconversion of a cyanobacterial photoreceptor, PixJ1, in phycocyanobilin-producing Escherichia coli

PixJ1, a photoreceptor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, mediates positive phototactic motility and contains two GAF domains, the latter of which binds a bilin chromophore. Full-length PixJ1 expressed and purified from Synechocystis showed unique reversible photoconversion between a blue light-absorbing (Pb) form and a green light-absorbing (Pg) form (1) in contrast to the reversible phototransformation between the red light-absorbing form and far-red light-absorbing form of the other GAF-containing photoreceptors such as plant or bacterial phytochromes. To clarify the origin of the blue-shifted photoconversion, we tried to reconstitute this blue-green reversible phototransformation by synthesizing the second GAF domain in Escherichia coli transformed with genes for biosynthesis of four different bilins, biliverdin (BV), bilirubin (BR), phycocyanobilin (PCB), and phycocyanorubin (PCR), as final products. The three expression systems, the BR system being the exception, produced a GAF polypeptide with a covalently bound bilin. The GAF polypeptide from the BV-synthesizing system exhibited an irreversible photoconversion, while that from the PCB-synthesizing system revealed photoconversion between Pb and Pg almost identical to that of the full-length PixJ1, indicating that PCB is responsible for the blue-green reversible photoconversion. Furthermore, the GAF polypeptide from the PCR-producing system exhibited almost the same reversible spectral change, possibly coming from the PCB accumulated in the PCR-biosynthetic pathway. Mass spectrometry (MS) of the main tryptic chromopeptide revealed that the chromophore binds to a 21-amino acid peptide that contains a cysteine-histidine motif for phytochrome chromophore binding and that an ion signal can be assigned to desorbed PCB. The absorption spectra of the denatured GAF polypeptide suggested that PCB is attached to the protein moiety in a twisted conformation that disrupts the pi-electron conjugation between the A and B rings, possibly being held in position through a second covalent linkage.     

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。     

Assembly of synthetic locked phycocyanobilin derivatives with phytochrome in vitro and in vivo in Ceratodon purpureus and Arabidopsis

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red light-absorbing form, Pr, and the far-red-light-absorbing form, Pfr. Here we performed in vitro and in vivo studies using locked phycocyanobilin derivatives, termed 15 Z anti phycocyanobilin (15ZaPCB) and 15 E anti PCB (15EaPCB). Recombinant bacterial and plant phytochromes incorporated either chromophore in a noncovalent or covalent manner. All adducts were photoinactive. The absorption spectra of the 15ZaPCB and 15EaPCB adducts were comparable with those of the Pr and Pfr form, respectively. Feeding of 15EaPCB, but not 15ZaPCB, to protonemal filaments of the moss Ceratodon purpureus resulted in increased chlorophyll accumulation, modulation of gravitropism, and induction of side branches in darkness. The effect of locked chromophores on phytochrome responses, such as induction of seed germination, inhibition of hypocotyl elongation, induction of cotyledon opening, randomization of gravitropism, and gene regulation, were investigated in wild-type Arabidopsis thaliana and the phytochrome-chromophore-deficient long hypocotyl mutant hy1. All phytochrome responses were induced in darkness by 15EaPCB, not only in the mutant but also in the wild type. These studies show that the 15Ea stereochemistry of the chromophore results in the formation of active Pfr-like phytochrome in the cell. Locked chromophores might be used to investigate phytochrome responses in many other organisms without the need to isolate mutants. The induction of phytochrome responses in the hy1 mutant by 15EaPCB were however less efficient than by red light irradiation given to biliverdin-rescued seeds or seedlings.     

Mechanism of Cph1 phytochrome assembly from stopped-flow kinetics and circular dichroism

The kinetics and mechanism of the autocatalytic assembly of holo-Cph1 phytochrome (from Synechocystis) from the apoprotein and the bilin chromophores phycocyanobilin (PCB) and phycoerythrobilin (PEB) were investigated by stopped flow and circular dichroism. At 1:1 stoichiometry, pH 7.9, and 10 degrees C, SVD analysis of the kinetic data for PCB revealed three spectral components involving three transitions with time constants tau(1) approximately 150 ms, tau(2) approximately 2.5 s, and tau(3) approximately 50 s. Tau(1) was associated with a major red shift and transfer of oscillator strength from the Soret region to the 680 nm region. When the sulfhydryl group of cysteine 259 was blocked with iodoacetamide, preventing the formation of a covalent adduct, a noncovalent red-shifted complex (680 nm) was formed with a time constant of 200 ms. Tau(1) could thus be assigned to the formation of a noncovalent complex. The absorption changes during tau(1) are due to the formation of the extended conformation of the linear tetrapyrrole and to its protonation in the binding pocket. From the concentration and pH dependence of the kinetics we obtained a value of 1.5 microM for the K(D) of this noncovalent complex and a value of 8.4 for the pK(a) of the proton donor. The tau(2) component was associated with a blue shift of about 25 nm and was attributed to the formation of the covalent bond (P(r)), accompanied with the loss of the 3-3' double bond to ring A. Tau(3) was due to photoconversion to P(fr). For PEB, which is not photochromic, the formation of the noncovalent complex is faster (tau(1) = 70 ms), but the covalent bond formation is about 80 times slower (tau(2) = 200 s) than with the natural chromophore PCB. The CD spectra of the PCB adduct in the 250-800 nm range show that the chromophore geometries in P(r) and P(fr) are similar to those in plant phytochrome. The opposite rotational strengths of P(r) and P(fr) in the longest wavelength band suggest that the photoisomerization induces a reversal of the chirality. The Cph1 complex with noncovalently bound PCB was still photochromic when cysteine 259 was blocked with IAA or with the bulkier IAF. The covalent linkage to cysteine 259 is thus not required for photoconversion. The CD spectra of the noncovalently bound PCB in P(r)- and P(fr)-like states are qualitatively similar to those of the covalent adducts, suggesting analogous structures in the binding pocket. The noncovalent interactions with the binding pocket are apparently sufficient to hold the chromophore in the appropriate geometry for photoisomerization.     

Chromophore composition of the phycobiliprotein Cr-PC577 from the cryptophyte Hemiselmis pacifica

The cryptophyte phycocyanin Cr-PC577 from Hemiselmis pacifica is a close relative of Cr-PC612 found in Hemiselmis virescens and Hemiselmis tepida. The two biliproteins differ in that Cr-PC577 lacks the major peak at around 612 nm in the absorption spectrum. Cr-PC577 was thus purified and characterized with respect to its bilin chromophore composition. Like other cryptophyte phycobiliproteins, Cr-PC577 is an (αβ)(α′β) heterodimer with phycocyanobilin (PCB) bound to the α-subunits. While one chromophore of the β-subunit is also PCB, mass spectrometry identified an additional chromophore with a mass of 585 Da at position β-Cys-158. This mass can be attributed to either a dihydrobiliverdin (DHBV), mesobiliverdin (MBV), or bilin584 chromophore. The doubly linked bilin at position β-Cys-50 and β-Cys-61 could not be identified unequivocally but shares spectral features with DHBV. We found that Cr-PC577 possesses a novel chromophore composition with at least two different chromophores bound to the β-subunit. Overall, our data contribute to a better understanding of cryptophyte phycobiliproteins and furthermore raise the question on the biosynthetic pathway of cryptophyte chromophores.     

Phytochrome assembly. Defining chromophore structural requirements for covalent attachment and photoreversibility.

Assembly of holophytochrome in the plant cell requires covalent attachment of the linear tetrapyrrole chromophore precursor, phytochromobilin, to a unique cysteine in the nascent apoprotein. In this investigation we compare chromophore analogs with the natural chromophore precursor for their ability to attach covalently to recombinant oat apophytochrome and to form photoactive holoproteins. Ethylidene-containing analogs readily form covalent adducts with apophytochrome, whereas chromophores lacking this double bond are poor substrates for attachment. Kinetic measurements establish that although the chromophore binding site on apophytochrome is best tailored to phytochromobilin, apophytochrome will accommodate the two analogs with modified D-rings, phycocyanobilin and phycoerythrobilin. The phycocyanobilin-apophytochrome adduct is photoactive and undergoes a light-induced protein conformational change similar to the native holoprotein. By contrast, the phycoerythrobilin adduct is locked into a photochemically inactive protein conformation that is similar to the red light-absorbing Pr form of phytochrome. These results support the hypothesis that the photoconversion from Pr to Pfr, the far red light- absorbing form of phytochrome, involves the photoisomerization of the C15 double bond. Knowledge gained from these studies provides impetus for rational design of chromophore analogs whose insertion into apophytochrome should elicit profound changes in light-mediated plant growth and development.     

Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481

When grown in green light, Fremyella diplosiphon strain UTEX 481 produces the red-colored protein phycoerythrin (PE) to maximize photosynthetic light harvesting. PE is composed of two subunits, CpeA and CpeB, which carry two and three phycoerythrobilin (PEB) chromophores, respectively, that are attached to specific Cys residues via thioether linkages. Specific bilin lyases are hypothesized to catalyze each PEB ligation. Using a heterologous, coexpression system in Escherichia coli, the PEB ligation activities of putative lyase subunits CpeY, CpeZ, and CpeS were tested on the CpeA and CpeB subunits from F. diplosiphon. Purified His(6)-tagged CpeA, obtained by coexpressing cpeA, cpeYZ, and the genes for PEB synthesis, had absorbance and fluorescence emission maxima at 566 and 574 nm, respectively. CpeY alone, but not CpeZ, could ligate PEB to CpeA, but the yield of CpeA-PEB was lower than achieved with CpeY and CpeZ together. Studies with site-specific variants of CpeA(C82S and C139S), together with mass spectrometric analysis of trypsin-digested CpeA-PEB, revealed that CpeY/CpeZ attached PEB at Cys(82) of CpeA. The CpeS bilin lyase ligated PEB at both Cys(82) and Cys(139) of CpeA but very inefficiently; the yield of PEB ligated at Cys(82) was much lower than observed with CpeY or CpeY/CpeZ. However, CpeS efficiently attached PEB to Cys(80) of CpeB but neither CpeY, CpeZ, nor CpeY/CpeZ could ligate PEB to CpeB.     

The complete amino-acid sequence of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon

The amino-acid sequences of both subunits of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon have been determined. The alpha-subunit contains 164 amino acid residues, two phycoerythrobilin (PEB) chromophores and has a molecular mass of 18,368 Da (protein: 17,192 Da + 2 PEB, one PEB accounting for 588 Da). The beta-subunit consists of 184 residues, three PEB chromophores and has a molecular mass of 20,931 Da (protein: 19,168 Da and 3 PEB: 1,764 Da). The five PEB chromophores (open chain tetrapyrroles) are covalently bound to six cysteine residues (one of them doubly bound to two cysteine residues). On the alpha-subunit, the first chromophore was found at position 84, homologous to the chromophore binding site of the other biliproteins APC, PC and PEC. The second chromophore, unique for the alpha-subunit of PE, is inserted together with a pentapeptide at position 143 a. On the beta-subunit, a doubly bound chromophore is attached to cysteine residues 50 and 61, similar to the rhodophytan phycoerythrins (B-PE and R-PE). The second and third chromophores were found at positions 84 and 155, homologous to the other biliproteins. A unique peptide insertion of 14 amino acid residues (without chromophore) was found at position 141 a-o in the beta-subunit and probably is located in the three-dimensional model near the additional chromophores of the C-PE alpha- and beta-subunits. Both additional chromophores of the C-PE alpha- and beta-subunit may be located at the periphery of the C-PE-trimer. The amino-acid sequence homology between C-PE alpha- and beta-subunit is 26% and to the alpha- and beta-subunits of C-PC from Mastigocladus laminosus 49% and 48%, respectively.