The Chromophorylation And The Structure Of Cyanobacteriochrome With PEB

Acted as photoreceptors in vivo, phycobiliproteins are the chromoprotein coupled with phycobilins. Phycobiliprotein photoreceptors can be divided into two categories including the antenna phycobiliproteins and photosensitive phycobiliprotein. Antenna phycobiliproteins capture and transfer energy from light to the photosynthesis system, while light-sensitive phycobiliproteins, also referred to as photochrome proteins, convert optical signals into biochemical signals, called phytochrome proteins. Antenna complexes of cyanobacteria phycobiliprotein are characterized with high fluorescence, but they also possess quite obvious flaws that they require participation of specific pigment synthetic enzymes and phycobiliprotein lyase to assemble the fluorescent markers. In recent years, some progress has been made in studies of cyanobacterial phytochrome. Conservative GAF domains are capable of binding and autocatalyzing a variety of pigments, with B V, PCB, PVB or even PEB, PUB included. The feature of GAF domains ensure that they have significant advantages over phycobiliproteins used as gene encoded fluorescent probe, to which largely enhancing the prospect of present chromoproteins’application.Several chromoproteins which do not exist in cyanobacteria were synthesized in our laboratory. Although they are not available in natural cyanobacteria, they could be generated by binding different pigment with apoproteins, which displays colossal potential in development of application. With this way of reconstitution, products people need can be selected and synthesized in a specific direction to be utilized as superior fluorescent labeling materials, such as fluorescent proteins with higher fluorescence quantum yield or the broader spectrum.In this study,16GAFs from three algae species, namely, Nostoc sp. PCC7120, Synechocystis sp. PCC6803and Thermosynechococcus elongatus BP-1were adopted to co-express with PEB chromophore synthase gene in E. coli in order to generate chromoprotein PEB-GAF or PUB-GAF, nine of which could autocatalyze chromophore to bind PEB, engendering intense fluorescence as bright as GFP. When GAFs were chromophorylated with PEB, three captivating fluorescent proteins were generated embracing PEB-A112699GAF1, PUB (PEB)-A113691GAF2, and PEB (PUB)-A111280GAF2. The maximum fluorescent peak of PEB-A112699GAF1shows at the wavelength of586nm, emitting orange fluorescence, whereas PUB (PEB)-A113691GAF2at498nm.(PUB)-A111280GAF2and PEB (PUB)-Sh1393GAF3PEB fluorescence emit green fluorescence at575nm with stokes shift of60-80nm and orange fluorescent emission. GAFs in combination with isomerized PEB can be used as green, orange, red fluorescent marker. GAFs chromophorylated by PEB has pros as follow:(1) GAF is a small unit to couple with chromophore, which does not demand the catalytic lyase.(2) PEB-chromophorylated GAFs have large molar extinction coefficient and high fluorescence quantum yield and brightness.(3) Some GAFs motif containing DXCF can isomerize PEB into PUB, so increasing the diversity of the PEB (PUB)-GAFs spectrum.Transformed PEB-chromophorylated GAFs into E.coli to express by inducement, bright fluorescent cells can be observed under fluorescent microscope.Although GAF can auto-catalyze binding of pigment, it still needs to coexpress with chromophore synthase gene. On the other hand, GFP, as a fluorescent protein, does not require other cofactors. Therefore, in order to simplify the phytochrome synthesis pathway, the method that links top and tail to gradually fuse genes is taken in this paper. First fuse hol and pebS into a gene encoded frame hoi: pebS, then insert the gaf domain into5’terminus of the fusion gene hoi: pebS. After the fusion gene gaf: hoi: pebS is constructed into the expression vector, it may be directly induced to express in host cells. In this paper, gene gafl encoded by all2699’s GAF domain and gaf2encoded by all1280’s domain from Nostoc sp. PCC7120as well as gene slr393’s gaf3from Synechocystis sp. PCC6803, producing chromoprotein PEB-GAF::HO1::PebS or PUB-GAF::HO1::PebS were fused with PEB synthase gene ho1:pebS, and then transformed into E.coli to express by inducement. Spectra show spectral characteristics consistent with the one before being simplified. These phytochrome complexes designed molecularly have the potential to become live cell fluorescence biomarkers. The chromophorylated GAFs then achieve its oligomerization via GCN4leucine zipper domains, to enhance their molar extinction coefficient and fluorescence quantum yield. PEB can be partially converted to PUB, making oligomeric GAF "bundle" form an interesting energy transfer model similar to that in light-harvesting antenna complexes in cyanobacteria. With benign thermal and photochemical stability and strong fluorescence intensity, it may have the very potential to become fluorescent immunoassay label.These results can be confirmed through spectroscopy, fluorescence microscopy. Superior spectral properties of chromophore fluorescent proteins are excellent supplement of GFP family fluorescent proteins, especially its orange-red fluorescence will provide convenience for tissue depth imaging, superresolution microscopy, and even three-dimensional data storage technology.In order to further molecular optimization of chromophore fluorescent proteins designed through molecule design, this study, based on trimming and cloning of gene fragments, successfully attained the efficient expression of recombinant PEB-GAF complex. It was identified by experiments that it maintains a correct spectral activity, and the crystal structure of PEB-A112699GAF1was successfully resolved at a resolution of1.7A using the GAF homology domain that had already been resolved in PDB database. Based on the structural analysis, a series of site-directed mutagenesis studies were conducted, three activity related mutant were designed, constructed and expressed, combined with related physiological experiment analysis to attempt to identify the active site and monomer of GAF domain structure.Studying through the combination of molecular design and structural biology not only has important theory significance, but has critical application prospect as well.