嗜热蓝藻层理鞭枝藻（Mastigocladus laminosus）藻胆体在解离过程中荧光发射光谱和光能传递的研究

研究了层理鞭枝藻藻胆体在不同浓度磷酸缓冲溶液中解离过程中荧光发射光谱的变化和光能传递。完整藻胆体的７７Ｋ荧光光谱中只有一个峰,位于６８５ｎｍ它是末端发射体（核心－膜连接多肽和别藻蓝蛋白－Ｂ）的荧光峰。部分解离藻胆体的荧光光谱的主峰位移至６５２ｎｍ：次峰位于６８５ｎｍ;６６０ｎｍ为一弱荧光发射肩。它们依次为Ｃ－藻蓝蛋白,末端发射体和别藻蓝蛋白的荧光。严重解离藻胆体的荧光主峰移６４４ｎｍ;次峰由６８５ｎｍ移至６８２ｎｍ;６６０ｎｍ荧光发射肩消失。这表明Ｃ－藻蓝蛋白所捕获的光能已不能传递给别藻蓝蛋白,但可传递给末端发射体洞时又表明Ｃ－藻蓝蛋白不仅与别藻蓝蛋白相连接而且还与末端发射体相连接。提出该藻胆体光能传递链如下：核心－膜连接多肽藻红蓝蛋白→Ｃ－藻蓝蛋白→别藻蓝蛋白别藻蓝蛋白－Ｂ     

嗜热蓝藻层理鞭枝藻（Mastigocladus laminosus）藻胆体在解离过程中荧光发射光谱和光能传递的研究

研究了层理鞭枝藻胆体在不同浓度磷酸冲溶液中解离过程中荧光发射光谱的变化和光能传递,完整藻胆体的７７Ｋ荧光光谱中只有一个峰,位于６８５ｎｍ,它是末端发射体（核心－膜连接多肽和别蓝蛋白－Ｂ）的荧光峰,部分解离藻胆体的荧光光谱的主峰位移至６５２ｎｍ,次峰位于６８５ｎｍ;６６０ｎｍ为一弱荧光发射肩,它们依次为Ｃ－藻蓝蛋白,末端发射体和别藻蓝蛋白的荧光。严重解离藻胆体的荧光主峰移至６４４ｎｍ;次峰由６８５ｎ     

R—藻红蛋白三维结构研究

藻类的捕光系统是由棒状的藻胆体构成的,藻胆体又是由藻红蛋白、藻蓝蛋白和变藻蓝蛋白组成的。光能从藻红蛋白传递到藻蓝蛋白,再传递到变藻蓝蛋白,最后传递到光反应中心,其传递效率接近１００％。Ｒ－灌红蛋是藻红蛋白一种,它是多亚基,超大分子量的蛋白色素复合物。为了阐明光能传递的机理,我们使用Ｘ－射线晶体学的方法,对取自多管藻的Ｒ－藻红蛋白的三维结构进行了长期的研究并取得了一系列进展。首先,我们使用多对同晶转     

增强型绿色荧光蛋白在集胞藻6803中的表达

利用聚球藻7942热休克基因groESL的启动子和报告基因egfp,构建了表达载体pUC-Tegfp并转化集胞藻6803,并通过所制备抗体对转基因藻进行蛋白免疫印迹检测.结果发现,在转基因藻株T-egfp的细胞粗提液中含有能与eGFP抗体特异结合的蛋白质,表明外源增强型绿色荧光蛋白基因(egfp)在集胞藻6803中成功表达.     

藻胆蛋白研究

藻胆蛋白是大量出现于红藻 (Ｒｈｏｄｏｐｈｙ ｔａ)、蓝绿藻 (Ｃｙａｎｏｐｈｙｔａ)和隐藻 (Ｃｒｙｐｔｏｐｈｙｔａ)中的捕光色素蛋白 ,主要包括藻红蛋白、藻蓝蛋白和别藻蓝蛋白三种。藻胆蛋白把捕获的光能高效地传递给叶绿素 ,从而使海藻的光合作用得以发生[1] 。细菌、藻类和高等植物的光合作用的共同特征是具有很多“天线分子” ,这些“天线分子”吸收光能并通过非放射性过程将激发能传递到含有叶绿素的“反应中心” ,在红藻、蓝绿藻和隐藻中 ,藻胆蛋白就充当这种“天线分子”的角色。因此 ,最初的藻胆蛋白研究主要集中在探讨其光合作…     

优化培养基增加层理鞭枝藻藻胆蛋白产量

藻胆蛋白(phycobiliprotein)是蓝藻和红藻藻胆体的组成部分,是光合作用集光复合体的组成部分,一般由α和β亚基构成,每个亚基含1～4个辅基色素,从而使藻胆蛋白具有特定的光谱吸收性质。根据这些吸收光谱性质,可以将藻胆蛋白分为:别藻蓝蛋白(APC)、藻蓝蛋白(PC)和藻红蛋白(PE)等,在某些缺乏PE而有异形胞的蓝藻中存在充当PE天线捕光功能的藻红蓝蛋白(PEC)〔1〕。藻胆蛋白可用于天然食用色素、化妆品色素和制药行业,还可作为免疫检测、荧光显微技术和流式细胞荧光测定法技术方面的荧光探针。特别是本工作研究的层理鞭枝藻(简称M.laminosu…     

螺旋藻（Spirulina platensis）藻胆体在解离过程中荧光发射光谱和光能传递的研究

对螺旋藻（Ｓｐｉｒｕｌｉｎａｐｌａｔｅｎｓｉｓ）藻胆体在室温和７７Ｋ处于不同浓度磷缓冲溶液和不同解离时间的荧光发射光谱进行了研究。藻胆体在０．９ｍｏｌ／Ｌ磷酸缓冲溶液中,由于没有发生解离,光能传递效率高,在７７Ｋ荧光发射光谱中只有一个峰,位于６８７ｎｍ,属于别藻蓝蛋白－Ｂ。当藻胆体悬浮在０．３ｍｏｌ／Ｌ磷酸缓冲溶液中１分钟,７７Ｋ荧光光谱的主峰出现在６８４ｎｍ．又出现６５５ｎｍ和６６６ｎｍ荧光峰,它们依次属子Ｃ－藻蓝蛋白和别藻蓝蛋白。在２小时;６５５ｎｍ荧先峰成为主峰,６８４ｎｍ荧光峰为次峰,６６６ｎｍ荧光肩消失。这表明Ｃ－藻蓝蛋白所捕获的先能已不能传递给别藻蓝蛋白,但能传给别藻蓝蛋白－Ｂ。我们提出在螺旋藻藻胆体中存在两类Ｃ－藻蓝蛋白,一是与别藻蓝蛋白相连接,另一是与别藻蓝蛋白－Ｂ相连接。     

螺旋藻（Spirulina platensis）藻胆体在解离过程中荧光发射光谱和光能传递的研究

对螺旋藻（Ｓｐｉｒｕｌｉｎａｐｌａｔｅｎｓｉｓ）藻胆体在室温和７７Ｋ处于不同浓度磷缓冲溶液和不同解离时间的荧光发射光谱进行了研究。藻胆体在０．９ｍｏｌ／Ｌ磷酸缓冲溶液中,由于没有发生解离,光能传递效率高,在７７Ｋ荧光发射光谱中只有一个峰,位于６８７ｎｍ,属于别藻蓝蛋白－Ｂ。当藻胆体悬浮在０．３ｍｏｌ／Ｌ磷酸缓冲溶液中１分钟,７７Ｋ荧光光谱的主峰出现在６８４ｎｍ．又出现６５５ｎｍ和６６６ｎｍ荧光峰,它们依次属子Ｃ－藻蓝蛋白和别藻蓝蛋白。在２小时;６５５ｎｍ荧先峰成为主峰,６８４ｎｍ荧光峰为次峰,６６６ｎｍ荧光肩消失。这表明Ｃ－藻蓝蛋白所捕获的先能已不能传递给别藻蓝蛋白,但能传给别藻蓝蛋白－Ｂ。我们提出在螺旋藻藻胆体中存在两类Ｃ－藻蓝蛋白,一是与别藻蓝蛋白相连接,另一是与别藻蓝蛋白－Ｂ相连接。     

R─藻红蛋白三维结构研究

藻类的捕光系统是由棒状的藻胆体构成的,藻胆体又是由藻红蛋白、藻蓝蛋白和变藻蓝蛋白组成的。光能从藻红蛋白传递到藻蓝蛋白,再传递到变藻蓝蛋白,最后传递到光反应中心,其传递效率接近100％。R─藻红蛋白是藻红蛋白的一种;它是多亚基,超大分子量的蛋白色素复合物。为了阐明光能传递的机理,我们使用X─射线晶体学的方法,对取自多管藻的R─藻红蛋白的三维结构进行了长期的研究并取得了一系列进展。首先,我们使用多对同晶置换法获得了R─藻红蛋白5A分辨率的结构,随后经过努力又获得了R─藻红蛋白2．8A中分辨率和1．9A高分辨率的结构,并首次解析了藻尿胆素的三维结构。我们通过对R─藻红蛋白三维结构的详细分析,对光能传递中的一些重要问题提出了自己的看法。该结构为我国有关藻类捕光蛋白的系列研究和光合作用机制研究打下了非常重要的基础。     

螺旋藻(Spirulina Platensis)藻胆体的光谱特性和能量传递的研究

研究了螺旋藻藻胆体的吸收光谱,室温和液氮温度荧光发射光谱和激发光谱.完整藻胆体的室温荧光峰位于678nm,不完整藻胆体位于672nm.在完整藻胆体的液氮温度荧光光谱中只有一个发射峰,不完整藻胆作有两个峰.研究结果表明C-藻蓝蛋白与别藻蓝蛋白之间的连接和别藻蓝蛋白与别藻蓝蛋白-B之间的连接具有不同的稳定性;前者稳定性较差,易解离.对藻胆体内藻胆蛋白之间的光能传递进行了讨论.     

集胞藻6803NdhO蛋白多克隆抗体制备及其初步应用

蓝藻NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体,参与CO2吸收、围绕光系统I的循环电子传递和细胞呼吸.迄今为止,人们在蓝藻细胞中已鉴定出17种NDH-1复合体亚基(NdhA-NdhQ).最近,人们还获得了NdhO亚基的缺失突变株.然而,人们对NdhO亚基的研究还不充份,至今仍不清楚它的功能角色.通过PC...     

Heterologous assembly and rescue of stranded phycocyanin subunits by expression of a foreign cpcBA operon in Synechocystis sp. strain 6803.

Light harvesting in cyanobacteria is performed by the biliproteins, which are organized into membrane-associated complexes called phycobilisomes. Most phycobilisomes have a core substructure that is composed of the allophycocyanin biliproteins and is energetically linked to chlorophyll in the photosynthetic membrane. Rod substructures are attached to the phycobilisome cores and contain phycocyanin and sometimes phycoerythrin. The different biliproteins have discrete absorbance and fluorescence maxima that overlap in an energy transfer pathway that terminates with chlorophyll. A phycocyanin-minus mutant in the cyanobacterium Synechocystis sp. strain 6803 (strain 4R) has been shown to have a nonsense mutation in the cpcB gene encoding the phycocyanin beta subunit. We have expressed a foreign phycocyanin operon from Synechocystis sp. strain 6701 in the 4R strain and complemented the phycocyanin-minus phenotype. Complementation occurs because the foreign phycocyanin alpha and beta subunits assemble with endogenous phycobilisome components. The phycocyanin alpha subunit that is normally absent in the 4R strain can be rescued by heterologous assembly as well. Expression of the Synechocystis sp. strain 6701 cpcBA operon in the wild-type Synechocystis sp. strain 6803 was also examined and showed that the foreign phycocyanin can compete with the endogenous protein for assembly into phycobilisomes.     

Mechanism of the down regulation of photosynthesis by blue light in the Cyanobacterium synechocystis sp. PCC 6803

Exposure to blue light has previously been shown to induce the reversible quenching of fluorescence in cyanobacteria, indicative of a photoprotective mechanism responsible for the down regulation of photosynthesis. We have investigated the molecular mechanism behind fluorescence quenching by characterizing changes in excitation energy transfer through the phycobilin pigments of the phycobilisome to chlorophyll with steady-state and time-resolved fluorescence excitation and emission spectroscopy. Quenching was investigated in both a photosystem II-less mutant, and DCMU-poisoned wild-type Synechocystis sp. PCC 6803. The action spectra for blue-light-induced quenching was identical in both cell types and was dominated by a band in the blue region, peaking at 480 nm. Fluorescence quenching and its dark recovery was inhibited by the protein cross-linking agent glutaraldehyde, which could maintain cells in either the quenched or the unquenched state. We found that high phosphate concentrations that inhibit phycobilisome mobility and the regulation of energy transfer by the light-state transition did not affect blue-light-induced fluorescence quenching. Both room temperature and 77 K fluorescence emission spectra revealed that fluorescence quenching was associated with phycobilin emission. Quenching was characterized by a decrease in the emission of allophycocyanin and long wavelength phycobilisome terminal emitters relative to that of phycocyanin. A global analysis of the room-temperature fluorescence decay kinetics revealed that phycocyanin and photosystem I decay components were unaffected by quenching, whereas the decay components originating from allophycocyanin and phycobilisome terminal emitters were altered. Our data support a regulatory mechanism involving a protein conformational change and/or change in protein-protein interaction which quenches excitation energy at the core of the phycobilisome.     

Picosecond kinetics of light harvesting and photoprotective quenching in wild-type and mutant phycobilisomes isolated from the cyanobacterium Synechocystis PCC 6803

In high light conditions, cyanobacteria dissipate excess absorbed energy as heat in the light-harvesting phycobilisomes (PBs) to protect the photosynthetic system against photodamage. This process requires the binding of the red active form of the Orange Carotenoid Protein (OCP(r)), which can effectively quench the excited state of one of the allophycocyanin bilins. Recently, an in vitro reconstitution system was developed using isolated OCP and isolated PBs from Synechocystis PCC 6803. Here we have used spectrally resolved picosecond fluorescence to study wild-type and two mutated PBs. The results demonstrate that the quenching for all types of PBs takes place on an allophycocyanin bilin emitting at 660 nm (APC(Q)(660)) with a molecular quenching rate that is faster than (1 ps)(-1). Moreover, it is concluded that both the mechanism and the site of quenching are the same in vitro and in vivo. Thus, utilization of the in vitro system should make it possible in the future to elucidate whether the quenching is caused by charge transfer between APC(Q)(660) and OCP or by excitation energy transfer from APC(Q)(660) to the S(1) state of the carotenoid--a distinction that is very hard, if not impossible, to make in vivo.     

The structure of monoacylglycerol lipase from Bacillus sp. H257 reveals unexpected conservation of the cap architecture between bacterial and human enzymes

In order to prevent photodestruction by high light, photosynthetic organisms have evolved a number of mechanisms, known as non-photochemical quenching (NPQ), that deactivate the excited states of light harvesting pigments. Here we investigate the NPQ mechanism in the cyanobacterium Synechocystis sp. PCC 6803 mutant deficient in both photosystems. Using non-linear laser fluorimetry, we have determined molecular photophysical characteristics of phycocyanin and spectrally distinct forms of allophycocyanin for the cells in non-quenched and quenched states. Our analysis of non-linear fluorescence characteristics revealed that NPQ activation leads to an ~2-fold decrease in the relaxation times of both allophycocyanin fluorescence components, F660 and F680, and a 5-fold decrease in the effective excitation cross-section of F680, suggesting an emergence of a pathway of energy dissipation for both types of allophycocyanin. In contrast, NPQ does not affect the rates of singlet-singlet exciton annihilation. This indicates that, upon NPQ activation, the excess excitation energy is transferred from allophycocyanins to quencher molecules (presumably 3'hydroxyechinenone in the orange carotenoid protein), rather than being dissipated due to conformational changes of chromophores within the phycobilisome core. Kinetic measurements of fluorescence quenching in the Synechocystis mutant revealed the presence of several stages in NPQ development, as previously observed in the wild type. However, the lack of photosystems in the mutant enhanced the magnitude of NPQ as compared to the wild type, and allowed us to better characterize this process. Our results suggest a more complex kinetics of the NPQ process, thus clarifying a multistep model for the formation of the quenching center.     

Distinct roles of CpcG1 and CpcG2 in phycobilisome assembly in the cyanobacterium Synechocystis sp. PCC 6803

Structural role of the second copy of the rod–core linker CpcG, which was found by genome analysis, was studied in Synechocystis sp. PCC 6803 by gene disruption and fractionation of phycobilisome (sub)complexes. Disruption of cpcG2 (sll1471) resulted in a marked decrease in phycocyanin content both in the background of wild-type and cpcG1 (slr2051)-disruptant. The unique phycocyanin rod–CpcG2 complex without the major allophycocyanin components was isolated from the cpcG1-disruptant. By fluorescence analysis, it was proposed that CpcG2 protein connects the rods with a minor allophycocyanin component, to support energy transfer to Photosystem I.     

Ssr2998 of Synechocystis sp. PCC 6803 is involved in regulation of cyanobacterial electron transport and associated with the cytochrome b6f complex

To analyze the function of a protein encoded by the open reading frame ssr2998 in Synechocystis sp. PCC 6803, the corresponding gene was disrupted, and the generated mutant strain was analyzed. Loss of the 7.2-kDa protein severely reduced the growth of Synechocystis, especially under high light conditions, and appeared to impair the function of the cytochrome b6 f complex. This resulted in slower electron donation to cytochrome f and photosystem 1 and, concomitantly, over-reduction of the plastoquinone pool, which in turn had an impact on the photosystem 1 to photosystem 2 stoichiometry and state transition. Furthermore, a 7.2-kDa protein, encoded by the open reading frame ssr2998, was co-isolated with the cytochrome b6 f complex from the cyanobacterium Synechocystis sp. PCC 6803. ssr2998 seems to be structurally and functionally associated with the cytochrome b6 f complex from Synechocystis, and the protein could be involved in regulation of electron transfer processes in Synechocystis sp. PCC 6803.     

Excitation energy transfer from phycocyanin to chlorophyll in an apcA-defective mutant of Synechocystis sp. PCC 6803.

A greenish mutant of the normally blue-green cyanobacterium Synechocystis sp. PCC 6803, designated UV6p, has been isolated and characterized. UV6p possesses functional photosystems I and II (PSI and PSII) but lacks normal light harvesting phycobilisomes because allophycocyanin is absent and core-specific linker proteins are almost entirely absent. The mutation responsible for the UV6p phenotype has been identified; it is a base substitution which results in the creation of a termination codon within the coding region of the apcA gene. Phycocyanin (PC) and phycobilisome rod linker proteins are present in UV6p and, despite the absence of core components, at least 35% of the PC is associated with rod linker proteins. At 77 K, light absorbed by PC of UV6p elicits PSI fluorescence comparable to that of wild type cells but produces greatly diminished PSII fluorescence. The results indicate that the assembly of rods is independent of cores and that light energy absorbed by rods can be transferred principally and directly to PSI. This energy transfer pathway, which may also be present in wild type, may have a regulatory role in maintaining the balance of input of excitation energy into PSI versus PSII during photosynthesis.     

Localization and function of ferredoxin:NADP(+) reductase bound to the phycobilisomes of Synechocystis.

Each phycobilisome complex of the cyanobacterium Synechocystis PCC 6803 binds approximately 2.4 copies of ferredoxin:NADP(+) reductase (FNR). A mutant of this strain that carries an N-terminally truncated version of the petH gene, lacking the 9 kDa domain of FNR that is homologous to the phycocyanin-associated linker polypeptide CpcD, assembles phycobilisome complexes that do not contain FNR. Phycobilisome complexes, consisting of the allophycocyanin core and only the core-proximal phycocyanin hexamers from mutant R20, do contain a full complement of FNR. Therefore, the binding site of FNR in the phycobilisomes is not the core-distal binding site that is occupied by CpcD, but in the core-proximal phycocyanin hexamer. Phycobilisome complexes of a mutant expressing a fusion protein of the N-terminal domain of FNR and green fluorescent protein (GFP) contain this fusion protein in tightly bound form. Calculations of the fluorescence resonance energy transfer (FRET) characteristics between GFP and acceptors in the phycobilisome complex indicate that their donor-acceptor distance is between 3 and 7 nm. Fluorescence spectroscopy at 77K and measurements in intact cells of accumulated levels of P700(+) indicate that the presence of FNR in the phycobilisome complexes does not influence the distribution of excitation energy of phycobilisome-absorbed light between photosystem II and photosystem I, and also does not affect the occurrence of 'light-state transitions'.