【作者】 孙雅芳；

【导师】 赵开弘；

【作者基本信息】 华中农业大学 ， 微生物学， 2014， 博士

【摘要】 藻胆蛋白是偶联藻胆色素的色素蛋白,在生物体内充当光受体。藻胆蛋白光受体可分为两类：天线藻胆蛋白、光敏藻胆蛋白。天线藻胆蛋白捕获光能并传递能量给光合作用系统,而光敏藻胆蛋白将光信号转化为生物化学信号,称为光敏色素蛋白。蓝细菌藻胆蛋白的天线复合物显示出高荧光特性,但也有明显的缺陷,在使其用做于良好的荧光标记物时,需要特定的色素合成酶以及藻胆蛋白裂合酶参与。近年,蓝细菌光敏色素的研究已有一些进展。保守的GAF结构域能自催化结合多种色素,包括BV、PCB、PVB甚至PEB、PUB。这一优良特性比藻胆蛋白用作可基因编码的荧光探针具有更多优点,大大增强了当前色素蛋白用作荧光探针的应用潜力。本实验室重组合成了一些在蓝藻体内并不存在的色素蛋白。这些色素蛋白虽然在天然蓝藻体内并不存在,但却可在体外利用不同色素与脱辅基蛋白组合生成,这在应用开发上具有很大潜力。利用这种重组方式,可定向选择合成人们需要的产物,如：荧光量子产率更高或者光谱范围更广的色素荧光蛋白,将他们用作优良的荧光标记材料。本论文尝试了来自三个藻种Nostoc sp. PCC7120、Synechocystis sp. PCC6803以及Thermosynechococcus elongatus BP-1中的16个GAF,分别与PEB色素合成酶基因在大肠杆菌体内共表达,产生色素蛋白PEB-GAF或PUB-GAF。其中9个可在大肠杆菌体内自催化色素化结合上PEB,并产生如荧光蛋白GFP一样的高荧光。将GAFs用PEB色素化,得到四个十分有趣的荧光蛋白：PEB-A11699GAF1、PUB(PEB)-A113691GAF2、 PEB(PUB)-All1280GAF2。 PEB-All21699GAF1最大荧光峰在586nm,发射橙红色荧光。PUB(PEB)-All3691GAF2最大荧光峰在498nm,发射绿色荧光。PEB(PUB)-A111280GAF2与PEB(PUB)-Slr1393GAf3相同,荧光在575nm处,有60-80nm的斯托克斯位移,发射橙色荧光。结合色素PEB且发生色素异构化的GAFs,可用作绿色、橙色、红色荧光标记物。用PEB色素化的GAFs,有以下优势：(1)GAF是一个偶联色素的小单位,其不需要裂合酶的催化。(2)PEB色素化后的GAFs有大的摩尔消光系数和高的荧光量子产率及亮度。(3)某些GAFs含有DXCF基序可将PEB异构为PUB,增加了PEB(PUB)一GAFs光谱的多样性。将荧光蛋白转化进大肠杆菌体内诱导表达,通过荧光显微镜的观察,可见明亮的荧光细胞。尽管GAF可自催化结合色素,但仍需要与色素合成酶基因共表达,而GFP作为荧光蛋白,不需要其他的辅助因子。因此,为了简化光敏色素合成途径,本论文用首尾相连逐步融合基因的方法解决这个问题。本论文将ho1和pebS先融合成一个基因编码框hol.pebS,然后再将gaf插入融合基因hol.pebS的5’末端。构建的融合基因gaf:hol:pebS连入表达载体后可在宿主细胞中直接诱导表达。本论文将实验室已有的来自于Nostoc sp. PCC7120的all2699的GAF结构域编码基因gafl、all1280的gaf2以及来自Synechocystis sp. PCC6803中的基因slr1393的gaf3分别与PEB色素合成酶基因hol:pebS融合,转化进大肠杆菌体内诱导表达,产生了色素蛋白PEB-GAF::HO1::PebS或PUB-GAF::HO1::PebS。光谱分析表明,光谱特征与简化前基本一致。这些经过分子设计的光敏色素复合物成为极有潜力的活细胞荧光生物标记物。色素化的GAFs可以进一步通过GCN4亮氨酸拉链结构域实现其寡聚化,以增强它们的摩尔消光系数和荧光量子产率。PEB可部分地向PUB转化,使寡聚的GAF"束”形成类似蓝藻中的捕光天线复合物中有趣的能量转移模型。良好的热、光化学稳定性及其强烈的荧光亮度,可成为极有潜力的荧光免疫检测标记物。这些结果通过光谱分析、荧光显微镜技术得以证实。色素荧光蛋白优异的光谱性质是GFP家族荧光蛋白的很好补充,尤其是它具有的橙红色荧光将为组织深度成像,超分辨显微技术,甚至三维数据存储技术提供便利。为对经分子设计的色素荧光蛋白进行进一步的分子优化,本论文在基因片段修剪克隆的基础上,成功实现重组PEB-GAF复合物的高效表达,实验鉴定其保持有正确的光谱活性,运用PDB数据库中已解析的GAF同源结构域成功解析了PEB-All2699GAF1复合物的晶体结构分辨率达1.7A的晶体。在分析结构的基础上进行了系列定点突变究,设计、构建、表达了3个相关突变体,结合相关生理实验分析,试图鉴定其活性部位及实现GAF结构域的单体化。本项研究通过将分子设计与结构生物学有机结合,不仅具有重要的基础理论意义,而且具有重要的应用前景。更多还原

【Abstract】 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.更多还原