【作者】 苏海楠；

【导师】 张玉忠；

【作者基本信息】 山东大学 ， 微生物学， 2010， 博士

【摘要】 藻胆体是存在于蓝藻和红藻中的一类捕光复合物(light-harvesting complex),是蓝藻和红藻光系统最主要的捕光天线(antennae),由多种藻胆蛋白和连接蛋白组成。根据光谱性质,藻胆蛋白可以划分为三个大类：异藻蓝蛋白、藻蓝蛋白和藻红蛋白,此外在部分蓝藻中还存在藻红蓝蛋白。藻胆蛋白以共价键连接多个色素基团,这些色素基团使得藻胆蛋白具有十分优良的捕获和传递光能的能力。在蓝藻和红藻细胞中,水溶性的藻胆蛋白位于类囊体膜的表面,直接暴露于细胞质或者类囊体基质中。当细胞内环境发生波动的时候,藻胆蛋白将受到直接影响,其构象很容易发生扰动。对于藻胆蛋白来说,在构象发生一定程度变化的时侯仍能维持捕获和传递光能的能力,对蓝藻和红藻在逆境条件下的生存是非常重要的。很多种类藻胆蛋白的晶体结构已经得到了解析,这些晶体结构提供了藻胆蛋白在平衡态下的静态结构信息。而藻胆蛋白在生理条件下的溶液中具有功能活性的构象,包括在受到环境干扰而发生结构扰动时的构象,必然是一个动态的变化范围。晶体结构只是提供了藻胆蛋白众多可能的构象当中的一种静态信息。因而2009年Liu等提出了藻胆蛋白活性构象的概念(Liu et al.,2009),并且把对藻胆蛋白的晶体结构的分析与藻胆蛋白在溶液中活性构象的动态变化的研究相结合,研究了在溶液中具有功能活性的藻胆蛋白的结构与功能的动态变化关系。本文对来源于蓝藻和红藻的各种种类的藻胆蛋白的活性构象进行了系统研究,主要研究内容和结果如下：一、异藻蓝蛋白高效分离纯化方法的建立异藻蓝蛋白是存在于蓝藻和红藻中的一类色素蛋白,在生物技术领域尤其是作为荧光标记物方面有着非常广泛的应用和巨大的市场价值。但异藻蓝蛋白无论在蓝藻中还是在红藻中都是一种低丰度的蛋白,含量很低,迄今为止仍缺乏高效制备型的分离纯化技术。现已报道的纯化技术均无法进行大量、高纯度的异藻蓝蛋白提取。本论文首先建立了一套从钝顶螺旋藻中高效、快速、低成本制备型分离纯化异藻蓝蛋白的方法。粗提液首先经过预处理,然后使用羟基磷灰石富集后再提取的方法,就能非常方便的提取出大量的异藻蓝蛋白。这种方法不仅能够在短时间快速提取出大量的异藻蓝蛋白,而且得到的异藻蓝蛋白纯度系数(A650／A280)达到2.0。提取出的异藻蓝蛋白再通过一步离子交换层析进行进一步纯化,得到高纯度的异藻蓝蛋白,其纯度系数(A650／A280)达到5.0,大大超过了国际上常用的商品化异藻蓝蛋白纯度系数(A650／A280)4.6的纯度标准。二、钝顶螺旋藻异藻蓝蛋白活性构象问题的研究异藻蓝蛋白是藻胆体核心部分的主要组成成分,能够吸收光能并且将能量传递到类囊体膜上的光系统中,推动光合作用顺利进行。本工作首先从螺旋藻中分离纯化了异藻蓝蛋白,然后以其为实验材料对异藻蓝蛋白光谱性质受溶液pH变化的影响做了研究。以紫外-可见光吸收光谱表征异藻蓝蛋白对光能的吸收能力,发现异藻蓝蛋白在650nm处的最大吸收峰能够在pH 4到10的范围之内,其峰形和峰值都能保持比较稳定,而在酸性环境中pH降低到4以下65nm处光吸收则完全消失,吸收峰蓝移至620nm。碱性环境中pH升高到11时异藻蓝蛋白在650nm的特征吸收峰才完全消失。异藻蓝蛋白在pH4到10范围之内稳定的光吸收能力通过荧光激发光谱的检测也得到了印证。使用荧光发射光谱表征异藻蓝蛋白的光能传递能力,发现异藻蓝蛋白的特征发射光谱峰位于660nm处,而且在pH值4到10的范围之内能够稳定的维持其发射荧光的强度,说明其光能传递能力是稳定的。使用圆二色谱检测了异藻蓝蛋白二级结构变化,发现异藻蓝蛋白的主要二级结构是α-螺旋,但是α-螺旋的含量随着溶液环境pH值的变化而发生一定程度的变动。同时还通过紫外-可见光吸收光谱、荧光光谱和可见光区圆二色谱分析了异藻蓝蛋白聚集状态的改变,发现异藻蓝蛋白在pH从4到10的范围之内,能够稳定的维持住其具有活性功能的三聚体聚集形式。因此光谱分析结果表明异藻蓝蛋白在一定pH值范围内,其光能吸收和传递的能力保持在一个相对较稳定的范围,同时异藻蓝蛋白维持聚集体的形式,而其二级结构则会发生一定程度的扰动。在pH值比较极端的情况下,异藻蓝蛋白三聚体发生解聚,二级结构发生剧烈变化,其光能吸收和传递能力迅速被破坏。通过分析异藻蓝蛋白的晶体结构发现异藻蓝蛋白α亚基和β亚基直接相互作用面上有一些关键的相互作用位点。因此,可能是通过这些相互作用位点,异藻蓝蛋白能够在局部结构发生一定程度的构象变化的情况下维持其蛋白的基本架构稳定,从而保证了其生理功能的稳定。三、钝顶螺旋藻藻蓝蛋白活性构象问题的研究藻蓝蛋白是蓝藻藻胆体杆部的最主要组成成分,具有执行吸收光能并且将光能传递到藻胆体核心部位的重要功能。本论文选择钝顶螺旋藻作为实验材料,分离纯化了藻蓝蛋白,对藻蓝蛋白在不同溶液pH值影响下的结构与功能的动态变化进行了研究。对藻蓝蛋白的光能吸收能力(以紫外-可见光吸收光谱表征)和光能传递能力(以荧光光谱表征)进行了监测,并且研究了藻蓝蛋白在功能受到影响时其二级结构的动态变化(以圆二色谱表征)和聚集状态的变化(以可见光区圆二色谱进行表征)。藻蓝蛋白在中性溶液环境中吸收光谱的最大吸收峰位于620nm处,在酸性环境下藻蓝蛋白会呈现一个复杂的变化过程,最大吸收峰会从620 nm处蓝移到615nm处,并且通过二阶导数分析发现峰形发生了变化。但是可见光区的吸收峰面积没有减少反而有所增强。在pH值从3.5到10这一比较宽泛的范围内,藻蓝蛋白对光能的吸收能力保持稳定。通过对藻蓝蛋白荧光激发光谱的研究也发现藻蓝蛋白能够在pH3.5到10的范围之内保持稳定的光吸收能力。藻蓝蛋白的荧光发射光谱最大发射峰在639nm处,其荧光发射峰的峰位随pH的变化有微小移动,但是峰值变化较小,反应了其能量传递能力的稳定性。通过分析藻蓝蛋白可见光区圆二色谱647nm处肩峰的谱线变化,发现藻蓝蛋白在功能保持稳定的pH范围之内聚集状态也是保持稳定的。通过使用紫外区圆二色谱对藻蓝蛋白二级结构的动态变化过程进行分析,发现藻蓝蛋白中的二级结构以α-螺旋为主,在藻蓝蛋白功能和聚集状态保持稳定的pH值范围内,其二级结构中α-螺旋的含量有一定程度的波动。因此,对光谱结果的分析表明藻蓝蛋白在溶液环境发生一定程度扰动的时候,能够保持其天然聚集状态,同时其对光能的吸收和传递的能力能够在一定范围内保持相对稳定,而此时其二级结构却发生了一定程度的扰动。随后分析了藻蓝蛋白的晶体结构,重点研究了藻蓝蛋白分子内部的亚基之间维持聚集状态的相互作用面,发现在α亚基和β亚基之间相互接触的面上的一些相互作用区域中存在一些关键的作用位点。通过这些相互作用位点,藻蓝蛋白得以维持其稳定的聚集状态。因此在受到一定程度的环境干扰时,这些相互作用位点能够比较稳定的维持藻蓝蛋白的聚集状态,而此时在蛋白结构的一些非关键区域中,肽链的构象能够呈现一定的柔性变化,这种蛋白折叠方式维持了藻蓝蛋白生理功能的稳定。四、多管藻藻红蛋白活性构象问题及溶液酸碱度对其光谱性质的影响藻红蛋白位于藻胆体杆的最末端,是红藻中最具有代表性的一类藻胆蛋白。本论对多管藻的藻红蛋的活性构象和去折叠过程中的光谱性质进行了研究。研究发现藻红蛋白在pH值从3.5到10的范围内,藻红蛋白中色基的构象相对稳定,光吸收能力也较为稳定。藻红蛋白在pH值从3.5到10的范围内荧光发射能力也保持较为稳定,并且在此范围内,藻红蛋白的各种二级结构含量有小幅波动,但总体上以α-螺旋结构为主,基本没有β-片层结构,转角和无规则卷曲含量略有波动。在pH低于3.25的酸性环境中,芳香族氨基酸暴露出来。在碱性环境中pH升高到10之后,蛋白构象的改变引起色基构象发生一定程度的变化,在此过程中可能还涉及色基的去质子化过程。在pH值比较极端的条件下荧光发射能力消失。当pH偏酸(小于3.5)或偏碱(大于10)时,α-螺旋结构开始急剧减少,β-片层结构和无规则卷曲结构开始大量增加。在极端pH条件尤其是碱性条件下,藻红蛋白的藻尿胆素(PUB)色基受溶液环境变化的干扰要比藻红胆素(PEB)受到的影响小。通过分析推测藻红蛋白在二级结构发生一定程度扰动的时候,仍然能够稳定的保持其光能的吸收和传递的能力。为了解释这个问题本论文同时分析了藻红蛋白的晶体结构,发现与其它藻胆蛋白相似的是,藻红蛋白在其亚基之间的相互作用面上有一些有相互作用较强的位点,正是这些位点之间的相互作用,使得藻红蛋白分子能够比较稳定的维持其聚集状态。在外界环境对藻红蛋白分子产生干扰的时候,藻红蛋白执行功能的区域也能够维持一定的稳定性,使得其能量吸收和转移的能力得到维持。而其它非关键区域则表现出一定程度的柔性,在蛋白结构受到干扰的时候发生一定的柔性变化。通过对藻红蛋白在pH影响下的光谱动力学变化的分析,发现藻红蛋白内部能量转移并没有受到溶液pH值改变而导致的藻红蛋白变性的影响。但是极端溶液pH值不仅可以导致藻红蛋白完全变性,还会修饰藻红蛋白的光谱性质,而且在酸性和碱性条件下修饰的方式是不一样的。五、藻胆蛋白中芳香族氨基酸与色基之间的相互作用藻胆蛋白中富含芳香族氨基酸,芳香族氨基酸的荧光随着藻胆蛋白的去折叠过程而发生变化,使用芳香族氨基酸荧光跟踪监测了藻胆蛋白在pH介导下的去折叠过程,发现在对藻胆蛋白中的芳香族氨基酸进行激发时,在可见光区发现了藻胆素的发射荧光,说明在藻胆蛋白内部存在着由芳香族氨基酸向藻胆素传递能量的路线。研究还发现,在通过激发芳香族氨基酸间接激发异藻蓝蛋白的藻胆素时,异藻蓝蛋白的藻胆素的发射荧光呈现出与直接激发藻胆素时得到的发射荧光不同的状态。通过对比其它藻胆蛋白的间接激发藻胆素得到的荧光以及对各种藻胆蛋白藻胆素构象的分析,推测在异藻蓝蛋白中存在两种不同构象的藻蓝胆素,它们在受激时能够分别发射出660nm和639nm的荧光。并且本文对间接激发下639nm荧光的出现等问题进行了讨论。更多还原

【Abstract】 Phycobilisomes (PBSs) are the major light harvesting pigment-protein complexes in cyanobacteria and red algae, and they are composed of various phycobiliproteins (PBPs) and linker polypeptides. Based on spectra properties, phycobiliproteins are commonly divided into three main groups:allophycocyanin (APC), phycocyanin (PC) and phycoerythrin (PE). PBPs are covalently linked with several co-factors named phycobilins, which enable PBPs to harvest solar energy and transfer it.PBSs in cyanobacteria and red algae locate on the surface of thylakoid membrane, directly exposing to cytoplasm or plastid stroma. PBPs may be directly affected by changes in cytoplasm or plastid stroma. For the PBPs in cyanobacteria and red algae, it is crucial to maintain their light-harvesting and energy transfer ability with respect to certain conformational changes. Crystal structures of PBPs provide us with static information. For a better understanding of the protein conformation, crystallography information of the proteins of interest is required to be combined with active structural and functional investigations.This thesis discussed the active conformations of the PBPs from cyanobacteria and red algae. The main results are as follows.1. Efficient separation and purification of APC from Spirulina platens isAPC is the core component of a PBS. It plays important physiological role in cyanobacteria and red algae, and it is also widely used in biochemical techniques and food industry. Here we established a method for extracting APC from Spirulina platensis with high efficiency. After pretreating the crude extract, we used hydroxylapatite to enrich and extract APC from PBP solution, and large amount of APC could be separated from other PBPs. This extracting method could not only separate large amount of APC in short time, but also obtain high purity of APC. APC extracted from crude extract could be further purified with single step of ion-exchange chromatography to obtain APC with higher purity. 2. Research on the active conformation of the APC from Spirulina platensisWe used the APC purified from Spirulina platensis, and studied its spectra property variations in response to pH changes. Light-harvesting ability was monitored by absorption spectra. Energy transfer ability was monitored by fluorescence emission spectra. Secondary structure was monitored by circular dichroism (CD) spectra. At the meantime, we studied the aggregation state of APC by absorption spectra, fluorescence spectra and vis-CD spectra. By analysis of spectra properties, we found that APC showed good absorbance and fluorescence stability at varying pH, with only minor changes between pH 4-10. The trimeric structure of APC was maintained while local variations of protein peptides were also shown in response to the environmental disturbance. Beyond this pH range, secondary structure as well as overall conformation of APC dramatically changed, and the energy absorption and transfer ability were also disrupted. By analysis of APC crystal structure, we found some key amino acid residues on the interaction surfaces betweenαandβsubunits. The fundamental tertiary of APC structure was probably stabilized by these contacts contributed by specific protein residues, whereas there might be some local conformational variations in response to environmental changes. Thus the physiological stability of APC was maintained.3. Research on the active conformation of the C-phycocyanin from Spirulina platensisWe purified the C-phycocyanin (C-PC) from Spirulina platensis. Variations of solution pH were used to disturb C-PC structure, and dynamics of structural and functional changes of C-PC were monitored in response to different pH values. We studied the light-harvesting ability (revealed by absorption spectra) and energy transfer ability (revealed by fluorescence spectra). At the mean time we studied changes of secondary structure (revealed by CD spectra) and aggregation state (revealed by vis-CD spectra). By analysis of these results, we found that C-PC could maintain its aggregation state even when there were some disturbances in solution, but the secondary structure might be flexible in a certain distance. Subsequently we analyzed crystal structures of C-PC, especially the interaction surfaces which maintain the aggregation state of the protein. We found some key sites on the interaction surfaces. By the interaction of these key amino acid residues, the aggregation state of C-PC was stabilized. Thus it is suggested that when certain environmental disturbance happens, these key sites can stabilize the aggregation state of C-PC, but in other regions the conformation of polypeptide could be flexible in response to environmental changes. Thus, C-PC maintained its functional stability with respect to certain conformational variations.4. Investigations of active conformational variation and the pH sensitivity of R-phycoerythrin from Polysiphonia urceolataPE is the characteristic PBP in red algae. We purified R-phycoerythrin (R-PE) from Polysiphonia urceolata and studied its active conformational variations and pH sensitivity. We found that, between the range of pH 3.5-10, the conformation of chromophores in R-PE was relatively stable, which was revealed by the stable absorption and fluorescence spectra properties of R-PE. While in this pH range, there were some variations in the amounts of secondary structures, the major structure is always a-helix. No P-sheet structure was detected. Amounts ofβ-turn and random coil structure varied in small scale. In acidic environment lower than pH 3.25, conformations of aromatic amino acid residues were exposed to the solution. In basic solution with pH higher than 10, there were conformational changes as well as deprotonation process of the chromophore. Fluorescence properties of R-PE disappeared in extreme pH. When pH was lower than 3.5 or higher than 10, amounts of a-helix decreased dramatically, while amounts ofβ-sheet and random coil structures increased. We analyzed the dynamics of spectra properties in response to solution pH, and found that the energy transfer ability in the protein was not influenced. We also found that extreme pH could not only induce denaturation of R-PE but also modulate spectra properties of the chromophores although the way of modulating was different between acidic and basic environment. Therefore, R-PE could maintain its light-harvesting and energy transfer ability in spite of some conformational variations. Then we analyzed the crystal structure of R-PE. Similar to other PBPs, there were some amino acid residues with strong interactions on the interaction surfaces of R-PE subunits. With these interactions, R-PE maintained its aggregation state. When environment conditions changed, the key regions, which are responsible for functional performances, maintained the structures and stabilized the energy absorption and transfer abilities of R-PE, while other regions showed some flexibility in response to disturbance.5. Interaction of aromatic amino acids with chromophores in PBPsAromatic amino acids are rich in PBPs. Fluorescence from aromatic amino acids changes following the folding/unfolding process of proteins. We monitored pH induced unfolding process of APC by fluorescence from aromatic amino acids. At the meantime, we found that when aromatic amino acids were excited, fluorescence from phycobilins arose in visible region of the spectra, suggesting there is energy transfer pathway from aromatic amino acid residues to the chromophores. We also found that when phycobilins were excited indirectly, fluorescence from APC was different from that when phycobilins were excited directly. By comparison of indirectly excited fluorescence from different PBPs and analysis of phycobilin conformations, we suggest that there are two types of phycocyanobilins in APC, which can emit fluorescence at 660 nm and 639 nm respectively. The existence of 639 nm fluorescence and other questions are also discussed.更多还原