【作者】 刘鲁宁；

【导师】 张玉忠；

【作者基本信息】 山东大学 ， 微生物学， 2008， 博士

【摘要】 藻胆体（PBsomes）是蓝藻和红藻中主要的捕光天线复合物。藻胆体由亲水性的藻胆蛋白（PBPs）和连接蛋白聚集而成,作为外部的超分子天线复合物,结合在类囊体膜的基质表面。藻胆蛋白是圆盘状结构的超分子蛋白质,由具有开链四吡咯结构的发色团（bilins）与脱辅基蛋白共价交联而成,有序的构成藻胆体。藻胆蛋白一般可以分为四类:藻红蛋白（PEs）、藻蓝蛋白（PCs）、别藻蓝蛋白（APCs）和少数藻红蓝蛋白（PECs）。在没有与光合作用反应中心复合物结合的情况下,藻胆体自身具有很强的荧光性。太阳光最初被藻红蛋白的色素吸收(λ_max=545～565 nm),然后被依次传递到藻蓝蛋白(λ_max=620nm)和别藻蓝蛋白(λ_max=650 nm),最终高效的传递到光反应中心的叶绿素。本论文研究了红藻中藻红蛋白的基本性质和藻胆体光谱结构特性,并应用显微镜技术从纳米尺度上揭示了红藻光合膜的天然构象和光适应性,藻胆体在类囊体膜上的扩散动态学,以及从单分子水平上探索了藻胆体的荧光动态过程。另外,对光合细菌Rhodobacter sphaeroides的LH2在二维晶体上的排布进行了研究。光合作用简介光合作用是光合生物体将太阳能转化为生物能的一个重要的生物过程。这种高效的反应从光合反应中心的捕光天线进行光能吸收开始。本论文第一章对光合作用的生物学意义、多样性和进化做了一个简介,重点介绍了蓝藻和红藻中的光合作用,以及其光合作用元件,如藻胆体、光反应中心、细胞色素Cyt b₆f复合物和ATP合成酶。特别对蓝藻和红藻的捕光天线系统——藻胆体的研究进展进行了综述,介绍了藻胆体的组成、光谱特性、结构和其中蛋白质—蛋白质之间的相互作用。本章还讨论了不同类型的光合作用复合物之间的相互关系,以及整个类囊体膜系统的超分子构象和生理的光适应性。藻红蛋白的分离纯化藻红蛋白被广泛应用在食品、化妆品、免疫诊断和分析试剂中。本论文第二章介绍了高效分离和纯化多管藻R-藻红蛋白的一步色谱法。该方法包括硫酸铵分步沉淀、DEAE-Sepharose Fast Flow离子交换层析。与以前报道的离子交换层析法相比,我们首次采用pH梯度洗脱蛋白的方法。通过该方法,纯化后的藻胆蛋白溶液的光吸收比(A₅₆₅/A₂₈₀达到5.6,回收率高达67.33%。这种有效的分离手段大大减少了传统工艺的分离步骤,降低了分离过程中蛋白损失和变性的几率,因此可以达到一个很高的蛋白回收率。随后我们对R-藻红蛋白的光谱特性、亚基组成和对pH的稳定性进行了研究。R-藻红蛋白的吸收光谱呈三峰型,吸收峰分别位于565、539和498 nm,室温荧光发射峰位于580nm。非变性电泳和SDS电泳检测了分离蛋白的纯度。结果证明,该方法是一种有效分离纯化多管藻R-藻红蛋白的手段,为进一步研究R-藻红蛋白的结构与功能打下了基础。R-藻红蛋白的活性结构与功能X—射线晶体衍射技术可以对蛋白质肽链和氨基酸结构进行高分辨率的解析。但是这个技术对于研究不同环境条件下蛋白的结构和功能的稳定性有一定局限性。在建立了有效分离R-藻红蛋白技术的基础上,第三章研究了pH诱导的R-藻红蛋白结构和功能的动态学过程。采用吸收光谱、荧光光谱和圆二色谱对R-藻红蛋白的光谱和结构变化进行检测,结合对现有的晶体结构进行分析。结果表明,R-藻红蛋白在pH 3.5-10范围内光谱特性稳定,而在pH 5-9范围内结构相对稳定。对结构的分析有助于我们了解R-藻红蛋白亚基的组成规律。R-藻红蛋白的四级结构通过一些关键位点的相互作用而构成。脱辅基蛋白肽为色素基团提供了稳定的蛋白环境,有利于其生理的能量传递功能。蛋白肽链局部的柔性构象是应对外界环境变化的一种策略。试验进一步揭示带电氨基酸和芳香族氨基酸在R-藻红蛋白结构的重要作用。芳香族氨基酸,尤其是酪氨酸（Tyr）能够与相邻的色素相互作用,从而影响其能量传递。该研究方法将静态的晶体结构分析与动态的蛋白质活性结构和功能研究相结合,为今后研究蛋白质的结构和功能提供了新的思路。藻胆体的单颗粒结构分析电子显微镜已经被用于观察紫球藻藻胆体的结构。第四章介绍了电子显微镜结合单颗粒平均技术在紫球藻藻胆体的超分子结构上的首次应用。结果表明半椭球状的藻胆体具有相对柔性的空间结构。相比而言,藻胆体—类囊体膜上藻胆体的结构比分离的藻胆体结构更稳定,从而有利于半椭球状构象的空间观察,并建立了半椭球状藻胆体的三维结构模型。另外研究表明,在低光照条件下,藻胆体在类囊体膜上形成有序排列结构域,而在相对强的光照下,藻胆体成无规则排列,藻胆体密度降低。这是首次在分离到的类囊体膜上观察到不同的藻胆体排列方式。这些结构数据有利于分析红藻藻胆体与光系统Ⅱ,以及集胞藻（Synechocystis）PCC 6803突变体中半圆盘状藻胆体与光系统Ⅱ可能的结合方式。红藻类囊体膜的天然构象和动态学在超分子水平上,整个光合作用膜网络的构象决定了大量光合作用蛋白复合物的生理作用。到目前为止,对红藻的类囊体膜表面的天然构象研究还较少。在第五章中,我们首次利用原子力显微镜（AFM）来研究红藻紫球藻的天然类囊体膜的超分子构象。在AFM中,单个藻胆体在空间上呈现半椭球状。另外,在不同的光强条件下,藻胆体在天然类囊体膜上表现出不同的排列方式:无规则排列和成排排列。而在上述不同的情况下,藻胆体的排列都是十分紧密的。这种紧密的分布不仅决定了类囊体膜上藻胆体的排列方式以及类囊体膜内光系统的排列规律,同时也限制了藻胆体在类囊体膜表面大范围的横向扩散。光脱色荧光恢复技术研究藻胆体的运动过程藻胆体被认为在蓝藻的类囊体膜表面上是可以横向移动的。而结构观察表明,膜上蛋白的拥挤排列大大限制了藻胆体的快速移动。在第六章中,我们利用光脱色荧光恢复技术（fluorescence recovery after photobleaching,FRAP）来研究紫球藻的类囊体膜上藻胆体的动态运动过程。对天然细胞的研究结果证实了荧光恢复现象的存在,这与蓝藻的观察结果一致。但是在用戊二醛固定的细胞体内以及体外分离出的藻胆体内也同样观察到了荧光恢复现象,这证明了藻胆体在红藻类囊体膜上的紧密排布限制了其水平的扩散运动。我们在红藻细胞中所观察到的荧光恢复现象是由于被光淬灭的藻胆体的自身光物理原因造成的,而不是由藻胆体在类囊体膜上的快速扩散所产生。藻胆体在类囊体膜上的快速扩散被认为与光能在两个光系统之间的分配有关,因此在红藻中一定有其他的与藻胆体相关的机制来完成光能的重新分配过程。同时,检测了光淬灭过程中藻红蛋白的荧光动态过程。体内和群体试验结果表明FRAP中的淬灭荧光可以引起藻红蛋白的荧光增强。对藻胆体和细胞进行戊二醛固定的对比试验揭示了光淬灭可以导致藻红蛋白在藻胆体杆上的能量解偶联。该反应可以使部分藻胆体的荧光从藻红蛋白散发出来,从而可以解释FRAP中只有部分荧光可以恢复的现象。藻胆体的单分子光谱研究在群体试验结果的基础上,第三章利用单分子光谱首次对红藻紫球藻（Porphyridium cruentum）的藻胆体进行研究,通过同步检测完整藻胆体中藻红蛋白和整个藻胆体的荧光强度,实时观测藻胆体在强光下的荧光动态过程。结果表明强绿光可以诱导藻胆体的荧光降低,而藻红蛋白的荧光在光淬灭（photobleaching）初期荧光增强。这说明藻胆体中藻红蛋白与邻近的藻胆蛋白之间发生了能量的解偶联。藻红蛋白的荧光随即降低,说明当藻胆体内能量传递受影响后,藻红蛋白作为单个荧光元件被淬灭。相比较,戊二醛固定后的藻胆体以及缺少B-藻红蛋白而只含有b-藻红蛋白的突变型藻胆体都没有发生能量解偶联。因此,结果表明这种能量解偶联是特异的发生在藻胆体杆中B-藻红蛋白和b—藻红蛋白的连接位点。同时,这种能量解偶联被证明具有光强依赖性和氧依赖性。光合作用生物体己经进化出多种保护机制来避免强光对细胞体内的光损伤。实验证明,橙色类胡萝卜素蛋白（orange carotenoid protein,OCP）在蓝藻的光保护机制中起到了重要的作用。然而藻胆体自身在蓝藻和红藻中的光保护作用还不清楚。这种能量解偶联被看作是藻胆体为了避免光系统受到光损伤所产生的生理对应机制,并揭示了红藻中含有γsubunit的藻红蛋白的新的光保护功能。AFM研究LH2的排布与蓝藻和红藻中的藻胆体不同,光细菌Rhodobacter sphaeroides的光合捕光复合物2（LH2）位于光合膜的内部,将光能传递给捕光复合物1（LH1）和反应中心。采用显微镜和光镜技术研究光合膜的超分子结构,证明光合作用蛋白复合物紧密的排列在膜上,蛋白的紧密排布对光合作用的功能结构域的形成和膜弯曲起到了重要作用。为了深入研究这种紧密排布效应,第八章利用AFM观察了R.sphaeroides LH2的二维晶体膜。与之前报道的一到二种排列方式不同,我们在一次制备中共观察到七种不同的排布阵列,其中LH2与膜表面均有倾斜角度。虽然LH2在体外以单体形式存在,但是我们发现了两种新的二聚体的排列方式。在第一种二聚体中,两个LH2单体相内倾斜;在第二种二聚体中,单体相外倾斜。进一步研究表明,这两种排列方式与“Z”型排列方式相似,倾斜角度也相对一致。其中第二种二聚体构象可以带动脂双层进行弯曲,在R.sphaeroides体内构成弧形的光合膜结构。更多还原

【Abstract】 Phycobilisomes（PBsomes）are the major light-harvesting antennae complexes in cyanobacteria and red algae.They are aggregations of water-soluble phycobiliproteins（PBPs）and linker polypeptides,and serve as external antenna macrocomplexes associated to the stromal surfaces of thylakoid membranes.PBPs are a distinctively colored group of disk-shaped macromolecular proteins bearing covalently attached open-chain tetrapyrroles,known as phycobilins（bilins）,orderly assembled into PBsomes.Four spectral groups of PBPs are commonly identified: phycoerythrins（PEs）,phycocyanins（PCs）,allophycocyanins（APCs）and sometimes phycoerythrocyanins（PECs）.In the absence of photosynthetic reaction centers（RCs）, the PBsomes are highly fluorescent.Solar energy is initially absorbed by the pigments of PEs(λ_max=545～565 nm)and transferred by nonradiative transfer in turn via PCs (λ_max=620 nm),APCs(λ_max=650 nm),and eventually to chlorophylls（Chls）with a high efficiency.In this thesis,I will present the detailed investigations on the properties of PEs, the spectral feature and the topography of PBsomes,the supramolecular architecture and photoacclimation of entire photosynthetic membrane in red algae using microscopic imaging in nano scale,the diffusion dynamics of PBsomes upon the thylakoid membrane,and the fluorescence dynamics of PBsomes at single molecule level.In addition,the packing organization of LH2s,the light-harvesting complexes from photosynthetic bacterium Rhodobacter sphaeroides,in artificially created 2D crystals is characterized.Introduction of photosynthesisPhotosynthesis is an essential conversion of solar light to biological energy in photosynthetic organisms.This highly efficient process starts from the light capturing by light-harvesting antenna of photosynthetic RCs.In Chapter 1,I provide a general introduction about the biological roles,diversity and evolution of photosynthesis. Then I focus on the photosynthesis in cyanobacteria and red algae,and their photosynthetic elements including the PBsomes,PSs,Cyt b₆f complexes and ATPase. In particular,the studies of light-harvesting antenna complexes,the PBsomes,are overviewed,consisting of its components,spectral properties,structures,and protein-protein interactions.The interactions of individual photosynthetic complexes, as well as the supramolecular architecture and the physiological photoacclimation of the overall thylakoid membrane network are summarized.Isolation of pure PEsPEs have been widely used in food,cosmetics,immunodiagnostics and analytical reagents.An efficient one-step chromatography method for purification of R-PEs from Polysiphonia urceolata was described in Chapter 2.Pure R-PEs were obtained with an absorbance ratio A565/A280 of 5.6 and a high recovery yield of 67.33%using a DEAE-Sepharose Fast Flow chromatography with a gradient elution of pH,alternative to common gradient elution of ionic strength.Such an effective methodology greatly reduces the traditional processing steps as well as the possibility of protein loss and denaturation during the overall operation,and a high recovery could thus be obtained.The absorption spectrum of R-PE was characterized with three absorbance maxima at 565 nm,539 nm and 498 nm,respectively.The fluorescence emission spectrum at room temperature was measured to be 580 nm.The results of native-PAGE,and SDS-PAGE showed no contamination by other proteins in the PE solution,which suggests an efficient method for the separation and purification of R-PEs from P urceolata for further accurate analysis.Active conformation and function of R-PEsX-ray crystallography of proteins has revealed high-resolution peptide conformations and amino acid organizations.However,investigations on the structural and functional stability of proteins in response to the environmental variations are limited in terms of this methodology.On the basis of the previous developed separating methodology of R-PEs,in Chapter 3,we explore the pH-induced conformational and functional dynamics of R-PEs isolated from P urceolata.The spectroscopic and structural variations of R-PEs monitored by means of absorption,fluorescence and circular dichroism（CD）spectra are investigated, together with analysis of the crystal structure of R-PE.R-PEs present a spectroscopic stability in pH range between 3.5 and 10,and relative structural sensitivity in pH range between 5 and 9,in response to the pH variations.Structural analysis allows us to better understand the assembly pattem of R-PE complexes.The tertiary structure of R-PE hexamer is fixed by specific interactions between several key anchoring residues,providing a stable protein environment for the chromophores to perform physiological energy migration.Local flexibility of protein peptide arrangement is allowed in response to the environmental disturbance.Our data further reveal that the charged amino acids and aromatic amino acid residues are highly involved in the association of R-PE complex.More specifically,aromatic amino acids,especially Tyr residues,are found to be capable to modify the interprotein energy transfer by close contacts with neighboring chromophores.This study combining analysis on the available crystal structure with active structural and functional investigations will provide new insights into the conformation and function of protein of interest,in addition to R-PEs.Single-particle structural inspections on the PBsomesThe structure of PBsomes from Porphyridium cruentum has been studied before with electron microscopy（EM）.In Chapter 4,EM combining with single particle averaging was performed for the first time to investigate the supramolecular architecture of PBsomes from P.cruentum.Isolated PBsomes are found to have a relatively flexible conformation.In contrast,PBsome-thylakoid vesicles provide relatively uniform PBsome structure,and allow us to acquire a spatial view of hemiellipsoidal structure.A three-dimensional model of the hemiellipsoidal PBsome is proposed.Under low-light growth conditions,the PBsomes on the membrane are mostly arranged in ordered domains.Whereas at higher light intensities,the distribution of PBsomes is largely disordered.It is the first time to observe the variety of PBsome arrangements upon isolated thylakoid membranes.We suggest that one PBsome likely lines up with one PSII dimer in red algae under low-light conditions is hypothesized because the red algal PSⅡis enlarged by a possible membrane-bound peripheral antenna which is absent in cyanobacteria.Native architecture and dynamics of thylakoid membrane of red algaeThe architecture of the entire photosynthetic membrane network determines,at the supramolecular level,the physiological roles of the photosynthetic protein complexes.So far,a precise picture of the native configuration of red algal thylakoids is still lacking.In Chapter 5,we investigate the supramolecular architectures of native thylakoid membranes from red alga P.cruentum,for the first time,using atomic force microscopy（AFM）.The topography of individual PBsomes is characterized to be spatially hemiellipsoidal.Furthermore,the native organization of thylakoid membranes presented variable arrangements of PBsomes,either a random arrangement,or rather ordered arrays of PBsomes,depending on light conditions.In particular,PBsomes were organized crowdingly in both cases.The packing of PBsomes is studied to determine not only the organizations of PBsomes,but also those of PSs in the thylakoid membrane.Furthermore,such crowding effects may restrict the large-scale lateral mobility of PBsomes on the surface of thylakoids. The dynamics of PBsomes studied using fluorescence recovery after photobleaching（FRAP）The lateral mobility of PBsomes on the surface of thylakoid membranes in cyanobacteria has been proposed.However,the structural inspections imply that the rapid diffusion of PBsomes may be greatly inhibited upon the crowding membrane surface.In Chapter 6,we examine for the first time the dynamic of photosynthetic membrane in red alga P cruentum with FRAP.Our data obtained from native cell showed the existence of partial fluorescence recovery,similar to that visualized in cyanobacteria.However,FRAP also occurs in the glutaraldehyde（GA）-fixed cell in vivo and ensemble PBsomes in vitro.Therefore,FRAP of red algal cell is ascribed to an intrinsic photophysics of the bleached PBsomes in situ,rather than the rapid diffusion of PBsomes on thylakoids in vivo,which has been proposed to be involved in excitation energy redistribution between photosystemⅠ（PSI）and photosystemⅡ（PSⅡ）.There should be other mechanisms for the PBsomes-related energy redistribution in red algae.In addition,we selectively monitor the fluorescence of PE instead of that of the entire PBsome in FRAP.The results of in vivo and ensemble experiments show that the bleaching laser applied in FRAP could result in the fluorescence increase of PE.Furthermore,the comparative data from GA-treated PBsomes and cells elucidate the energetic decoupling of PEs in the PBsome rods.Due to this decoupling,part of the fluorescence of PBsomes is dissipated from PE.It can presumably explain the partial fluorescence recovery observed in FRAP.Single-molecule spectroscopic study on isolated PBsomesAccording to the ensemble results,in Chapter 7,single-molecule spectroscopy is applied for the first time on the PBsomes of red alga P.cruentum to detect the fluorescence emissions of PEs and PBsome terminal emitters（APB）simultaneously, and the real-time detection could greatly characterize the fluorescence dynamics of individual PBsomes in response to intense light.Our data reveal that strong green-light can induce the fluorescence decrease of APB,as well as the fluorescence increase of PE at the first stage of photobleaching.It strongly indicated an energetic decoupling occurring between PE and its neighbor.The fluorescence of PE was subsequently observed to decrease,showing that PE could be photobleached when energy transfer in the PBsomes was disrupted.In contrast,the energetic decoupling was not observed in either the PBsomes fixed with GA,or the mutant PBsomes lacking B-PE and remaining b-PE.It was concluded that the energetic decoupling of the PBsomes occurs at the specific association between B-PE and b-PE within the PBsome rod.In addition,this process is demonstrated to be power- and oxygen-dependent.Photosynthetic organisms have developed multiple protective mechanisms to prevent photodamage in vivo under high-light conditions.In cyanobacteria,the orange carotenoid protein（OCP）has been demonstrated to play roles in the photoprotective mechanism.However,the direct PBsome-related energy dissipation mechanism in red algae is still unclear.Such a decoupling process is proposed to be a strategy corresponding to the PBsomes to prevent photodamage of the photosynthetic RCs. Furthermore,our results implied a novel photoprotective role ofγ-subunit-containing PE in red algae.Packing of LH2s studied by AFMUnlike PBsomes in cyanobacteria and red algae,the peripheral photosynthetic LH2 complexes from the bacterium Rhodobacter sphaeroides are embedded in the photosynthetic membranes,transferring energy to LH1 and RCs.Microscopic and light spectroscopic investigations on the supramolecular architecture of bacterial photosynthetic membranes have revealed the photosynthetic protein-complexes to be arranged in a densely packed energy-transducing network.Protein packing may play a determinant role in the formation of functional photosynthetic domains and membrane curvature.To further investigate in detail the packing effects of like-protein photosynthetic complexes,in Chapter 8,I report an AFM investigation on artificially created 2D-crystals of LH2s from R.sphaeroides.Instead of the usually observed 1 or 2 different crystallization lattices for one specific preparation protocol we find 7 different packing lattices.The most abundant crystal types all show a tilting of the LH2 complex.Most surprisingly,although the LH2 complex is a monomeric protein-complex in vivo,we find a LH2 dimer packing motif.I further characterize two different dimer configurations:in Type 1 the LH2 complexes are tilted inwards,in Type 2 outwards.Closer inspection of the lattices surrounding the LH2 dimers indicates their close resemblance to those LH2 complexes that constitute a lattice of zig-zagging LH2.In addition,analyses of the tilt of the LH2 complexes within the zig-zag lattice and that observed within the dimers corroborate their similar packing-motif.The Type 2 dimer configuration exhibits a tilt that,in absence of up-down packing,could bend the lipid bi-layer leading to the strong curvature of the LH2 domains as observed in R.sphaeroides photosynthetic membranes in vivo.更多还原