蓝藻别藻蓝蛋白的生物合成及组装的研究

【作者】 刘少芳；

【导师】 秦松；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋药物学， 2010， 博士

【摘要】 蓝藻是地球上分布最广泛、最原始的放氧光合作用原核生物,其光合作用是从藻胆蛋白组成的藻胆体结构吸收光能开始的。藻胆蛋白是一个多亚基组成的蛋白复合物,每个亚基上结合不同的开环四吡咯色素分子(色基),使得它们可以吸收不同波长的光能。能量在藻胆蛋白之间几乎以100%的效率传递到光反应中心。别藻蓝蛋白(APC)位于藻胆体核心,具有特殊的光谱特征。APC由α和β亚基组成,每个亚基只结合一个色基PCB,但由单体(αβ)聚集为三聚体(αβ)3后,其光谱性质发生了巨大的变化。APC是以三聚体形式参与光能传递的,因此,研究其合成和组装能使我们更好的理解其功能,即光能传递的基础,并为人工组装藻胆体提供根据。本文以集胞藻PCC 6803 APC为研究对象,首先研究了APC的生物合成,即色基PCB是如何共价结合到脱辅基亚基上的过程。我们在大肠杆菌体内重组了荧光APCα和β亚基生物合成的完整通路。利用双表达载体,将APC脱辅基亚基ApcA或ApcB、合成色基的血红素氧化酶HO1、胆绿素还原酶PcyA及催化PCB结合到脱辅基亚基上的裂合酶CpcS/U在大肠杆菌中共同表达。通过诱导,大肠杆菌能够利用自身的亚铁血红素合成具有与天然APC亚基一致的特征吸收和荧光光谱的重组色素蛋白,holo-ApcA和holo-ApcB。研究还证实,只有在裂合酶CpcS/U同时表达时才能完成色基PCB与脱辅基亚基的正确连接。我们对APCα亚基与PCB进行了体外重组研究,并提出了裂合酶可能的作用机理。裂合酶CpcS/U之间存在相互作用,共同存在时使它们的溶解性和稳定提高。在体外实验中,裂合酶CpcS/U可以与色基迅速的以非共价方式结合,在APC脱辅基亚基存在的条件下,酶还可以将色基转移至脱辅亚基上,并帮助其以正确的构型与蛋白共价结合,获得与天然APC亚基一致的光谱特性。在体外条件下,脱辅亚基能与色素PCB自发结合,但该过程非常缓慢,并且无法获得正确的光谱特征。APC亚基的体内组建和体外重组实验为阐明APC在蓝藻中的生物合成过程提供了理论依据。在确定了集胞藻PCC 6803 APC亚基的生物合成途径的基础上,我们在大肠杆菌体内对APC组装为三聚体(αβ)3进行了研究。利用双表达载体,将APC脱辅基亚基ApcA和ApcB、裂合酶CpcS/U、色素合成酶Ho1和PcyA在大肠杆菌中共同表达。通过多步分离的方法,包括亲和色谱柱、分子排阻凝胶色谱柱等方法,获得与天然别藻蓝蛋白光谱一致的蛋白。根据光谱特征分析、荧光量子产率等显示了重组蛋白具有天然APC相似的性质。胰蛋白酶消化实验证实重组三聚体与天然APC三聚体水解色基多肽的HPLC图谱相近,可以证明利用大肠杆菌体内重组所得到的色素蛋白与天然APC色基结合位点一致。这是首次报道了在大肠杆菌体内重组的APCα和β亚基组装成具有天然APC结构特性的三聚体。以前有报道脱辅基亚基在大肠杆菌的共表达只能获得异二聚体,与该结果相比较,我们认为色基在三聚体组装中起重要作用。LCM是APC重要的连接蛋白,是藻胆体能量传递的终端受体,并对其组装和锚定起重要作用。LCM也是藻胆体中最大的色素分子,由于其本身溶解性很差,关于其生化性质和生物合成的研究不多。我们在大肠杆菌体内重组了LCM(1-240)生物合成的通路。LCM(1-240)在大肠杆菌体内主要以包涵体的形式存在,溶于4M尿素后获得与报道的LCM基本一致的光谱特性。为了提高其溶解性,得到天然状态的表达蛋白,在LCM(1-240)的N端引入了大肠杆菌麦芽糖结合蛋白MBP,得到了光谱特征一致的可溶性的LCM(1-240),这为下一步对其生化性质进行深入的研究提供了基础。对APC的生物合成及组装的研究,有助于我们探讨其结构组成对光谱特性的影响,理解其在蓝藻光传递系统的功能,并有利于进一步阐明别藻蓝蛋白多亚基组装的机理,为下一步人工合成藻胆体提供理论依据。另外,天然别藻蓝蛋白作为荧光标志物在生物学和临床领域应用广泛,通过重组方式获得别藻蓝蛋白,将可以使我们通过分子设计的方法获得功能改进的蛋白,具有一定的应用价值。更多还原

【Abstract】 Cyanobacterium is one of the primitive species with the capacity of oxygenic photosynthesis. The process of photosynthesis is initiated by the absorption of light energy through phycobiliproteins. Phycobiliproteins are multi-subunit complex that covalently bind a variety of linear tetrapyrolle pigments called bilins, enabling them to harvest light in the visible region of the spectrum. The absorbed energy can be transferred at almost 100% efficiency to the reaction center.Allophycocyanin (APC), located in the core of phycobilisome, has the special spectroscopic characteristics. APC is tightly associatedαβheterodimer, with only one bilin covalently attached to each subunit. One of the prominent spectroscopic characteristics of APC is the strong red-shift of the absorption and fluorescent emission maxima when monomers assembling into trimers. APC participates in the energy transfer in higher aggregates, i.e. APC trimer. Therefore, the researches on the biosynthesis and assembly of APC will provide insight into its function, i.e. the basis of the energy transfer. Further, these will also help us in the reconstitution of the giant complex phycobilisome.In this study, the entire pathway for the biosynthesis of holo-αandβsubunits of APC from Synechocystis sp. PCC 6803 was reconstituted in Escherichia coli (E. coli). The genes for apo-proteins (apo-ApcA or apo-ApcB) biosythesis, the genes ho1 and pcyA encoding the enzymes for PCB production and the genes cpeS-1 and cpcU for the attachment of PCB to apo-proteins were co-expressed in E. coli by a dual vector system. Upon induction, holo-αandβsubunits of APC were acquired with spectroscopic properties similar to those of the same protein produced in cyanobacteria. The results were also shown that CpcS/U is the bilin lyase which responsible for attachment of PCB to both ApcA and ApcB. In addition, the reconstitution of holo-ApcA was performed in vitro and the possible catalysis mechanism for the attachment of PCB to apo-ApcA was also suggested. The results demonstrated that CpcS/U can form a heterodimer so as to improve their solubility and stability. Lyases CpcS/U can bind rapidly to PCB in a non-covalent way, and transfer it to apo-ApcA slowly under the appropriated conditions. The reconstituted products have the characteristics identical to that of native APC holo-αsubunit. PCB can spontaneously attach to apo-ApcA in a much slower way, however, complex with correct absorption maxima could not be acquired in the absence of lyases CpcS/U. Both the reconstitution of APCsubunits in vivo in E. coli and in vitro will provide insight into the biosynthetic process of APC in cyanobacteria.In this study, the fluorescent APC trimer was successfully synthesized from Synechocystis sp. PCC 6803 in E. coli. Genes encoding APC apo-α,βsubunits and enzymes for phycocyanobilin (PCB) biosynthesis and covalent attachment to apo-proteins were co-expressed in E. coli using a dual plasmid system. The recombinant APC trimer (rAPC) was purified by using metal affinity and size exclusion chromatography and showed the characteristic absorption and emission spectra identical to those of the native APC, suggesting that rAPC was in its native conformation. Tryptic digestion analysis confirms the rAPC has the same components as the native APC. The molecular weight analysis (by HPLC) results clearly show that rAPC is in a trimeric state. This is the first study on the assembly of recombinant ApcA and ApcB to a trimer with the native structure.When compared to the study in which only monomer was formed, our results indicate that the synthesis of bilins and the subsequent attachment to apo-subunits are very important for the successful assembly of APC trimers.LCM is a multifunction linker serving as a terminal energy acceptor and a membrane anchor. Information on their biochemical properties and biosynthesis is scare due to their insolubility. In this study, the pathway for the synthesis of LCM(1-240) was reconstituted in E. coli. The recombinant LCM(1-240) is prone to aggregation as inclusive body. These insoluble products were dissolved in 4 M urea to acquire the spectroscopic properties similar to those of the reported proteins. The solubility of LCM(1-240) is improved when it is expressed as a MBP fusion, without the changes of the spectroscopic properties. This study will facilitate the biochemical analysis of LCM(1-240), and allow further understanding of their functions.Our studies focus on the biosynthesis and assembly of APC, which would help us to understand the relationship between the structure and the functions, reflecting on the change of its spectra. These would also facilitate the interaction analysis of multi-subunits of APC, which leads to the assembly of trimers. Additionally, as APC is a widely used fluorescent tags with numerous applications in biological and clinical fields, this study will provide a promising way to produce better fluorescent tags by molecular design.更多还原