Bacteriophytochromes (Bphs) comprise a family of protein photoreceptors that help bacteria sense changes in light. Bphs contain a chromophore that, upon absorption of red or far-red light, undergoes a cis-trans isomerization that leads to a conformational change in the holoprotein (photoconversion). In the active conformation, Bphs act as a kinase and regulate gene expression through phosphorylation of target proteins. Two putative Bph orfs (rpa0122 and rpa0990) in the Rhodopseudomonas palustris genome encode Bph-like proteins that have a conserved chromophore-binding cysteine residue. The hypothesis is that one or both of these unique Bph-like genes encode proteins that are capable of binding a chromophore and functioning to modulate the cell’s phenotype. I expressed and purified His-tagged RPA0990 in R. palustris, because proteolytic degradation was observed during overexpression in an E coli. expression system. The results show that RPA0990 contains a chromophore and is capable of photoconversion. The wavelengths of light absorbed by the Pr/Pfr forms of RPA0990, predicted to be active and inactive forms respectively, were determined to be 695 nm and 755 nm. Investigation into the phenotype of the bph mutants rpa0122 and rpa0990 revealed that both of these Bphs may have a small effect on light-harvesting complexes. Also, it was observed that the absence of O₂ does not inhibit the normal function of Bphs, although O₂ was thought to be needed to make a linear tetrapyrrole cofactor, by cleaving heme using heme oxygenase. I suggest that a linear tetrapyrrole can be made anaerobically, either through anaerobic heme cleavage by a novel enzyme, or directly from the heme precursor hydroxymethylbilane without ring cleavage. The activity of a divergent promoter region between the rpa1490 (bph3) and rpa1491 (pucBe) genes was evaluated by using the E. coli lacZ gene as a reporter. The results indicated that the pucBe promoter has much higher activity than the bph3 promoter. It was also found that double knockout of the regulatory genes ppsR1ˉ2ˉ led to an increase in bph3::lacZ expression and a decrease in pucBe::lacZ expression.

Bacteriophytochromes (Bphs) comprise a family of protein photoreceptors that help bacteria sense changes in light. Bphs contain a chromophore that, upon absorption of red or far-red light, undergoes a cis-trans isomerization that leads to a conformational change in the holoprotein (photoconversion). In the active conformation, Bphs act as a kinase and regulate gene expression through phosphorylation of target proteins. Two putative Bph orfs (rpa0122 and rpa0990) in the Rhodopseudomonas palustris genome encode Bph-like proteins that have a conserved chromophore-binding cysteine residue. The hypothesis is that one or both of these unique Bph-like genes encode proteins that are capable of binding a chromophore and functioning to modulate the cell’s phenotype. I expressed and purified His-tagged RPA0990 in R. palustris, because proteolytic degradation was observed during overexpression in an E coli. expression system. The results show that RPA0990 contains a chromophore and is capable of photoconversion. The wavelengths of light absorbed by the Pr/Pfr forms of RPA0990, predicted to be active and inactive forms respectively, were determined to be 695 nm and 755 nm. Investigation into the phenotype of the bph mutants rpa0122 and rpa0990 revealed that both of these Bphs may have a small effect on light-harvesting complexes. Also, it was observed that the absence of O₂ does not inhibit the normal function of Bphs, although O₂ was thought to be needed to make a linear tetrapyrrole cofactor, by cleaving heme using heme oxygenase. I suggest that a linear tetrapyrrole can be made anaerobically, either through anaerobic heme cleavage by a novel enzyme, or directly from the heme precursor hydroxymethylbilane without ring cleavage. The activity of a divergent promoter region between the rpa1490 (bph3) and rpa1491 (pucBe) genes was evaluated by using the E. coli lacZ gene as a reporter. The results indicated that the pucBe promoter has much higher activity than the bph3 promoter. It was also found that double knockout of the regulatory genes ppsR1ˉ2ˉ led to an increase in bph3::lacZ expression and a decrease in pucBe::lacZ expression.

Bacteriophytochromes (Bphs) comprise a family of protein photoreceptors that help bacteria sense changes in light. Bphs contain a chromophore that, upon absorption of red or far-red light, undergoes a cis-trans isomerization that leads to a conformational change in the holoprotein (photoconversion). In the active conformation, Bphs act as a kinase and regulate gene expression through phosphorylation of target proteins. Two putative Bph orfs (rpa0122 and rpa0990) in the Rhodopseudomonas palustris genome encode Bph-like proteins that have a conserved chromophore-binding cysteine residue. The hypothesis is that one or both of these unique Bph-like genes encode proteins that are capable of binding a chromophore and functioning to modulate the cell’s phenotype. I expressed and purified His-tagged RPA0990 in R. palustris, because proteolytic degradation was observed during overexpression in an E coli. expression system. The results show that RPA0990 contains a chromophore and is capable of photoconversion. The wavelengths of light absorbed by the Pr/Pfr forms of RPA0990, predicted to be active and inactive forms respectively, were determined to be 695 nm and 755 nm. Investigation into the phenotype of the bph mutants rpa0122 and rpa0990 revealed that both of these Bphs may have a small effect on light-harvesting complexes. Also, it was observed that the absence of O₂ does not inhibit the normal function of Bphs, although O₂ was thought to be needed to make a linear tetrapyrrole cofactor, by cleaving heme using heme oxygenase. I suggest that a linear tetrapyrrole can be made anaerobically, either through anaerobic heme cleavage by a novel enzyme, or directly from the heme precursor hydroxymethylbilane without ring cleavage. The activity of a divergent promoter region between the rpa1490 (bph3) and rpa1491 (pucBe) genes was evaluated by using the E. coli lacZ gene as a reporter. The results indicated that the pucBe promoter has much higher activity than the bph3 promoter. It was also found that double knockout of the regulatory genes ppsR1ˉ2ˉ led to an increase in bph3::lacZ expression and a decrease in pucBe::lacZ expression. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Given a transfer function for a differential equation model, an approach for obtaining a solution is by way of bilinear transformation. The bilinear transform approach is a numerical integration scheme which gives a discrete approximation to the differential equation solution. BILIN applies a series of polynomial transformations to the transfer function H(s). As a result, H(s) is mapped into the complex z plane obtaining the discrete transfer function H(z). From H(z), the difference equation is obtained whose solution y(nT) approximates the actual differential solution y(t). Hence, BILIN provides a means for obtaining discrete transfer functions for the design of digital filters and/or solving linear time-invariant differential equations.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24997号 / 医博第5031号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 羽賀 博典, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The goal of this research is to identify and characterize enzymes involved in bilin attachment to the phycobiliprotein allophycocyanin in the cyanobacterium Synechococcus sp. PCC 7002. Candidates for lyases responsible for attachment of phycocyanobilin to allophycocyanin are two cpeS-like genes termed cpcS and cpcU, and one cpeT-like gene termed cpcT. In vitro bilin attachment reactions were conducted in the presence of the recombinant substrate apo-allophycocyanin (HT-ApcAB). Size exclusion HPLC showed that CpcS and HT-CpcU form a 1:1 heterodimeric complex and that HT-ApcAB is present as a monomer (áâ). Absorbance and fluorescence spectroscopy illustrated that both CpcS and HT-CpcU were required to get holo-allophycocyanin with phycocyanobilin attached to the cysteine-81 residue. Absorbance of the product at 615 nm was consistent with holo-monomeric allophycocyanin. Experiments were performed with HT-ApcD ApcB and HT-ApcF ApcA, but size exclusion HPLC showed they were in aggregated form.

The goal of this research is to investigate the role of the slr1098 gene in the cyanobacterium Synechocystis sp. PCC 6803, a gene with similarity to cpcE which encodes a subunit of an enzyme involved in bilin attachment to phycocyanin. This protein is hypothesized to be involved in oligomerization of phycocyanin due to previous results showing the mutant made shorter phycocyanin rods. The recombinant Slr1098 protein was produced and purified from E. coli cells. Binding assays showed interaction between Slr1098 and both apo- and holo-phycocyanin, but not to apo-allophycocyanin. Slr1098 blocked bilin addition at Cys-82 on CpcB by the CpcS/CpcU bilin lyase. Size exclusion chromatography and sucrose density gradient analysis of complexes formed suggest that Slr1098 strongly interacts with all intermediate forms of phycocyanin and may be an important checkpoint in the biosynthesis and oligomerization of this protein, but that by itself, Slr1098 does not increase oligomerization of phycocyanin.

Phytochromes (Phys) are biliproteins that regulate light responses in plants, fungi, and microorganisms through photoconversion between a dark state and a photoproduct. Thermal reversion of the photoproduct is an intrinsic property of all Phys, typically occurring on a timescale of seconds to days. Despite methodological advances, the structural and spectroscopic determination of short-lived photoproducts has proven challenging. We herein present an innovative approach for photoproduct stabilisation by incorporating the protein into trehalose glasses (TGs). The resulting Phy–trehalose matrices were investigated by UV/Vis absorption and solid-state NMR spectroscopies. Our results demonstrate that the TGs strongly inhibit thermal reversion of the incorporated Phy proteins for periods as long as several weeks at room temperature (RT), during which the proteins fully sustain their native structures and spectral and biochemical properties. This sample preparation approach is beneficial for revealing bona fide structure/ function relationships of short-lived photoproducts that are otherwise not accessible, thus paving the way towards a deeper molecular understanding of the diversified spectral properties of Phys. Our results also provide new insights into the molecular mechanism of trehalose bioprotection.

Cyanobacteria are photosynthetic prokaryotes that able to produce oxygen. They have light harvesting complexes called phycobilisomes (PBS). PBS are generally composed of an allophycocyanin core with phycocyanin and phycoerythrin rods connected to the core. PBS are able to efficiently harvest light energy from different wavelengths of visible light due to the evolution of PBP. Phycoerythrin has five chromophores that are attached to six cysteine residues and is essential for efficient green light capture and transfer of energy for use in photosynthesis. The attachment of these chromophores to PBP is facilitated by enzymes known as bilin lyases. In this study, we characterize and explore the role of enzymes that are involved in the biosynthesis of phycoerythrin in cyanobacteria. Biochemical and molecular techniques were used in the characterization of these proteins to gain a better understanding of their roles in the post-translational modification of phycobiliprotein. In F. diplosipohon, the lyase activity of CpeT was characterized and studied using a heterologous, co-expression system in E. coli. It was determined that CpeT was able to ligate PEB to Cys-165 of CpeB in the presence of CpeZ, a chaperone-like protein. Next, the roles of three proteins, MpeY from RS9916 and MpeQ and MpeW from A15-62, were analyzed using a combination of gene-interruption mutants and recombinant protein expression techniques. The absence of mpeY resulted in the reduction of PEB chromophorylation of MpeA in green light conditions, and recombinant protein coexpression confirmed that MpeY was responsible for PEB attachment to Cys-83 of MpeA. The interruption of mpeQ in A15-62 resulted in a reduced PUB phenotype in MpeA in blue light. Recombinant protein expressions revealed that MpeQ was a lyase-isomerase responsible for the attachment of PUB to Cys-83 of MpeA. Two regulatory proteins located in two conserved configurations of a genomic island present in species that are able to change their phycobilin content in response to different light environments, known as Type-IV chromatic acclimation (CA4), were investigated. FciA and FciB from RS9916 were studied using gene interruption mutants from RS9916 and they were found to be responsible for the CA4 response in CA4-A containing species of Synechococcus.

Phytochromes constitute one of the six well-characterized families of photosensory proteins in Nature. From the viewpoint of computational modeling, however, phytochromes have been the subject of much fewer studies than most other families of photosensory proteins, which is likely a consequence of relevant high-resolution structural data becoming available only in recent years. In this work, hybrid quantum mechanics/molecular mechanics (QM/MM) methods are used to calculate UV-vis absorption spectra of Deinococcus radiodurans bacteriophytochrome. We investigate how the choice of QM/MM methodology affects the resulting spectra and demonstrate that QM/MM methods can reproduce the experimental absorption maxima of both the Q and Soret bands with an accuracy of about 0.15 eV. Furthermore, we assess how the protein environment influences the intrinsic absorption of the bilin chromophore, with particular focus on the Q band underlying the primary photochemistry of phytochromes.