Reconstitution In Vivo And Photochemical Properties Of Cyanobacteriochrome Domains

Plant phytochromes, being a photoreceptor, that is a water-soluble proteins attach linear tetrapyrrole chromophore and exhibits reversible photoconversion. And we have known that phytochromes are also present in bacteria and fungi from recent studies. Cyanobacteriochromes （CBCRs） are a spectrally diverse photoreceptor family that binds a bilin chromophore. CBCRs are spectrally diverse covering through out the near-ultraviolet/visible light region, they are distantly related to phytochromes and have been found only in cyanobacteria. CBCRs may be involved in a light-induced reset of the circadian rhythm and cell aggregation. Experiment showed that, the separate GAF domains were cloned into expression vector, the strain harboring the expression plasmid and the plasmid for bilin chromophore were induced to generate recombinant proteins, photochemical properties of chromoproteins were similiar to CBCRs holoproteins.The cyanobacterium Thermosynechococcus elongatus BP-1is thermophilic, its CBCRs are relatively small in number and thermostable. By protein sequence homology comparison with the Pb/Pg-type CBCRs TePixJ and TeTlr0924, four homologous genes tll0899, tlr0911, tlr1215and tlr1999from T. elongatus BP-1were found. By molecular cloning techniques, the GAF domains of those genes were cloned into expression vector pET30a（+）, respectively. The E. coli strain BL21harboring the expression plasmid and the pACYCDuet-hol-pcyA plasmid were induced to generate recombinant proteins. The experimental results showed that Tl10899-GAF contained one covalently bound bilin chromophore, phycocyanobilin （PCB）; Tlr0911-GAF contained two covalently bound bilin chromophores, phycoviolobilin （PVB） and phycocyanobilin （PCB）, and exhibited reversible photoconversion between a blue-absorbing form at406nm （Pb406nm） and a green-absorbing form at527nm （Pg527nm）; Tlr1999-GAF was also covalently bound with PVB and PCB, and reversible photoconversion existed between a blue-absorbing form at417nm （Pb417nm） and a teal-absorbing form at496nm （Pt496nm）.Tlr0911-GAF and Tlr1999-GAF were denoted blue/green photoreceptors because their absorption covering through out the blue and green light region. The GAF domain of Tlr0911and Tlr1999 may be multifunctional by itself in vivo:it acts as an autolyase, a photoisomerase and in some instances as an autoisomerase. Neither Tlr1215-GAF1nor Tlr1215-GAF2could be spontaneously bound with bilin chromophore.In the study of the only two putative CBCRs Sync₀012and Sync₁604in Cyanobacteria Synechococcus sp. CC9311, by molecular cloning techniques, the GAF domains of the two genes were cloned into expression vector pET30a（+）, respectively. The expression plasmid in the E. coli strain BL21was alone induced expression for proteins, or both the expression plasmid and the pACYCDuet-hol-pcyA plasmid in the E. coli strain BL21were induced to generate recombinant proteins. The solubility of the proteins were quite low, and the proteins were solubilized by adding urea. Results showed, neither Sync₀012-GAF nor Sync₁604-GAF could be spontaneously bound with bilin chromophore, but they may be covalently bound with riboflavin from the E. Coli, and these required further research.According to amino acid sequence of GAF domains of putative CBCRs in Nostoc PCC7120for protein clustering analysis, using of biology softwares Clustal X1.83and MEGA version5.0, a phylogenetic tree was obtained. The phylogenetic tree indicated a photoreceptor branch that contained the conserved DXCF motif clearly distinct from other GAFs domains, as well as it contained some other photoreceptor branches. By molecular cloning techniques, the GAF domains of those genes were cloned into expression vector pET30a（+）, respectively. The E. coli strain BL21harboring the expression plasmid and the pACYCDuet-hol-pcyA plasmid were induced to generate recombinant proteins. The experimental results showed that DXCF CBCRs All1688and Alr1966had various reversible photoconversion. A111688-GAF was covalently bound with PCB, and contained a weakly reversible photochemistry. Absorption spectra of blue-absorbing state and green-absorbing state were largely overlap. Alr1966-GAF2was covalently bound with PVB and PCB, they included three absorbing states, and named Pg state, the intermediate state and Pb state. A sequential reversible photoconversion cycle was existed in the three absorbing state. Another photoreceptor branch was consisted of All4978-GAF, A117016-GAF and Alr7522-GAF, in which Alr7522-GAF did not have the photochemical activities, the remaining two proteins had similar photochemical properties, but they did not have a reversible photochemistry. In accordance with their absorption spectroscopy, we speculated that they may be combined with heme.By protein sequence similarity and homology comparison with Slr1393, S1ll041, UirS （gene named slr1212）, respectively, three homologous genes sll1888, sll1228and sll1124from Synechocystis sp. PCC6803were found. By molecular cloning techniques, the GAF domains of the three genes were cloned into expression vector pET30a（+）, respectively. The E. coli strain BL21harboring the expression plasmid and the plasmid pACYCDuet-hol-pcyA were induced to generate recombinant proteins. The experimental results showed that they could not be spontaneously bound with bilin chromophore and does not have photochemical properties.