应用飞秒时间分辨瞬态吸收光谱研究蛋白质中距离相关长程电荷转移:光合细菌天线复合体LH2与TiO2纳米颗粒超分子组装体个例初探

蛋白质在生物体内电荷转移过程中所起的作用迄今仍然是一个有争议的问题.其争论焦点是蛋白质在生物电荷转移过程中是否提供特殊的电子传递通道或者是仅仅作为普通的有机介质.应用飞秒时间分辨瞬态吸收光谱研究由光合细菌天线分子和平均粒径为8 nm的TiO2组装而成的超分子系统中长程电荷转移.晶体结构研究表明,光合细菌天线分子具有由多个α-脱辅基和β-脱辅基蛋白跨膜螺旋构成的双层空心柱面体结构,其中α-脱辅基蛋白跨膜螺旋构成的小环状体套于β-脱辅基蛋白跨膜螺旋构成的大环状体中.小环状体的空腔直径约为3.6 nm.光合色素细菌叶绿素和β-胡萝卜素分子处于两环之间.细菌叶绿素距离外周胞质膜最近,预计为1 nm.本研究试图将TiO2纳米颗粒部分装入光合细菌膜蛋白的腔体中,探讨细菌叶绿素与TiO2纳米颗粒间进行的光致长程电荷转移,进而揭示蛋白质在电荷转移过程中所起的作用.实验观察到细菌叶绿素B850在LH2/TiO2中的基态漂白恢复的时间常数明显地比在LH2中短,应用长程电荷转移模型,将蛋白质视为普通介电媒体,由电荷转移速率推算得到细菌叶绿素与TiO2纳米颗粒最近表面的距离为0.6 nm,表明TiO2纳米颗粒已经成功地部分装入光合细菌天线分子的空腔中.     

光系统Ⅱ反应中心CP47/D1/D2/Cyt b-559复合物中色素分子空间取向的线二色光谱研究

线二色光谱(LD)是研究色素分子在光合膜上空间取向和排布的重要手段.采用低温(100K)吸收光谱和线二色光谱技术研究光系统Ⅱ核心复合物CP47/D1/D2/Cyt b-559中色素分子的空间取向.结果表明,在光系统Ⅱ核心复合物CP47/D1/D2/Cyt b-559中680 nm处有吸收的叶绿素分子Qy跃迁与光合膜平面平行.β-胡萝卜素分子有两种不同的空间取向,其中在470和505nm处有吸收的β-胡萝卜素分子(Ⅰ)与光合膜平面近似平行,而在460和490nm处有吸收的β-胡萝卜素分子(Ⅱ)与光合膜垂直.光破坏实验显示垂直取向的β-胡萝卜素分子对强光敏感.680nm处吸收的叶绿素分子成分复杂,可能包含有P680和核心天线CP47蛋白上的色素分子.     

光系统Ⅱ反应中心CP47/D1/D2/Cyt b-559复合物中色素分子空间取向的线二色光谱研究(英文)

线二色光谱(LD)是研究色素分子在光合膜上空间取向和排布的重要手段。采用低温(1 0 0K)吸收光谱和线二色光谱技术研究光系统Ⅱ核心复合物CP47/D1/D2/Cytb_5 5 9中色素分子的空间取向。结果表明,在光系统Ⅱ核心复合物CP47/D1/D2/Cytb_5 5 9中 6 80nm处有吸收的叶绿素分子Qy 跃迁与光合膜平面平行。β_胡萝卜素分子有两种不同的空间取向,其中在 470和 5 0 5nm处有吸收的 β_胡萝卜素分子(Ⅰ)与光合膜平面近似平行,而在 46 0和 490nm处有吸收的 β_胡萝卜素分子(Ⅱ)与光合膜垂直。光破坏实验显示垂直取向的 β_胡萝卜素分子对强光敏感。6 80nm处吸收的叶绿素分子成分复杂,可能包含有P6 80和核心天线CP47蛋白上的色素分子。     

固氮红细菌(Rhodobacter azotoformans) 423 nm特征吸收峰成因与定位

【背景】在不产氧光合细菌中,因420-425nm特征峰位于类胡萝卜素(Carotenoid,Car)吸收区域,通常被认为是由Car积累引起,但固氮红细菌R7菌株呈现的423 nm特征峰不具备Car三指峰特征。【目的】阐明R7菌株423 nm特征吸收峰形成的物质基础及胞内定位。【方法】采用吸收光谱、薄层层析、高效液相色谱、质谱、超速离心和离子交换层析等方法阐明423 nm吸收峰形成原因。【结果】谷氨酸钠明显促进R7菌株活细胞呈现423 nm特征峰,色素提取液中该峰蓝移至415 nm,但其生长、细菌叶绿素(Bacteriochlorophyll,BChl)和Car含量大幅度降低,而添加酵母提取物则反之。色素组成分析表明,在检测到的色素成分中,只有镁卟啉单甲基酯Ⅸ (Magnesium Protoporphyrin Ⅸ Monomethylester,MPE)呈现415 nm特征吸收峰。MPE可定位于光合膜上并呈现出423 nm特征峰。对色素蛋白复合体(Pigment Protein Complex,PPC)的研究显示,添加谷氨酸和酵母提取物的菌体细胞虽然都检测到3种PPC组分[2个外周捕光复合体(Peripheral Light Harvesting Complex 2,LH2)和1个光反应中心(Reaction Center,RC)],但源自谷氨酸菌体细胞的RC和1个LH2则呈现423 nm特征吸收峰,表明R7菌株可产生2种不同类型的LH2,且MPE可定位于一种LH2和RC。【结论】R7菌株所呈现的423 nm特征峰不是由Car积累所致,而是由MPE积累所形成,且能与LH2和RC结合定位于光合膜上。MPE是BChl合成的中间产物,其合成受严格调控,不容易获得。MPE代谢调控的深入研究可为光合作用光氧化损伤与保护机理增添新内容。     

太阳能电池反应模拟光合系统反应中心电荷复合形成三线态分子的过程

光合系统反应中心普遍存在电荷复合反应形成三线态分子的过程,并通过所形成的三线态β-胡萝卜素将剩余的能量经无辐射通道耗散给环境,实现光合系统的光保护功能.这一过程在人工合成系统中十分罕见,见诸报道的仅有少数由给体-受体组成的超分子体系.首次报道应用染料敏化TiO2胶体颗粒的人工太阳能电池反应,模拟光合系统三线态分子的形成过程,成功地观测到了视黄酸自由基正离子与TiO2表面束缚电子复合而形成的三线态视黄酸分子,并对其光谱和动力学过程进行了纳秒时间分辨光谱表征.     

LH2中类胡萝卜素为链孢红素的类球红细菌绿色突变株(GM309)的获得及理化分析

用甲基磺酸乙酯诱变类球红细菌(Rhodobacter sphaeroides 601), 分离获得绿色突变株, 命名为GM309. 吸收光谱分析表明其外周天线色素复合物(LH2)的类胡萝卜素特征吸收峰发生了20 nm的蓝移. 通过吸收光谱和质谱进一步的分析确定RS601的LH2中类胡萝卜素为球形烯, 而GM309的LH2中则变为链孢红素. 链孢红素比球形烯少一个共轭双键. CD, 吸收光谱分析表明GM309 LH2中色素排列结构与野生LH2相近. 比较野生和突变株的LH2荧光发射光谱, 发现GM309的LH2仍保持了激发能由类胡萝卜素向细菌叶绿素以及细菌叶绿素B800向B850的传递, 其B850的荧光峰为对照的58%, 表明激发能由细菌叶绿素B800向B850的传递效率有所降低. 研究为了解类胡萝卜素在LH2激发能传递中的作用提供了新的实验手段.     

固氮红细菌(Rhodobacter azotoforman)色素蛋白复合体的分离纯化与特性

【目的】为揭示不产氧光合细菌产氢菌株色素蛋白复合体(PPC)色素组成和含量与光合放氢的关系奠定基础。【方法】以PPC特征光谱为检测指标,采用硫酸铵分级分离、DEAE-纤维素层析、吸收光谱和SDS-PAGE等方法进行了固氮红细菌(Rhodobacter azotoformans,R.azotoformans)R7产氢菌株PPC的分离纯化、纯度分析和鉴定;采用表面增强激光解吸电离离子飞行时间质谱、HPLC-MS和荧光光谱法对其中一种PPC进行了组成分析和能量传递活性测定。【结果】从R7菌株获得了3种纯化的PPC,1种为反应中心与中心捕光色素蛋白复合体(RC-LH1),2种为外周捕光色素蛋白复合体(LH2),其中一种LH2的吸收光谱具有异常的423nm强吸收峰,其蛋白的两种亚基的分子量分别为5356.8Da和5697.8Da,类胡萝卜素属球形烯系,分子量为562Da,激发光能够从类胡萝卜素向细菌叶绿素以及细菌叶绿素向细菌叶绿素传递。【结论】固氮红细菌产氢菌株含有2种不同光谱特性的LH2,其中一种具有新光谱特性。     

体外组装类胡萝卜素对外周捕光复合体(LH2)能量传递活性的影响

【目的】探求不产氧光合细菌(APB)外周捕光复合体(LH2)中类胡萝卜素(Car)结构和能量传递效率的关系和规律。【方法】通过二苯胺(DPA)抑制Car合成的方法从固氮红细菌134K20中获得部分缺失Car的LH2 (LC-LH2);采用TLC和HPLC法从3种APB中制备球形烯(SE)、玫红品(RP)和奥氏酮(OK) 3种Car;在含0.1%十二烷基二甲基胺氧化物(LDAO)的10 mmol/L Tris-HCl (pH 8.0)缓冲液中采用超声孵育法分别将这3种Car与LC-LH2体外组装,采用吸收光谱法、拉曼光谱法和荧光光谱法对组装LH2进行结构与功能分析。【结果】制备的部分缺失Car的LH2中,SE缺失率约为64.7%。这3种共轭长度、取代基的极性不同的Car均能与这种部分缺失SE的LH2自组装,Car组装率约在24.0%?29.4%之间,其中SE和OK的组装率高于RP。与部分缺失Car LH2中原有SE构象一致,重组的Car在LH2中也呈现较为伸展的平面构象。LH2中重组Car到细菌叶绿素(BChl)的能量传递效率由高到低的顺序依次为SE-LH2>RP-LH2>OK-LH2,与Car共轭体系大小的关系一致,而与Car极性大小没有明显的关系。【结论】在组装的LH2中Car采用平面构象与脱辅基蛋白结合,Car共轭长度仍是决定和影响LH2中Car-BChl能量传递效率的主要因素,而Car的取代基和极性影响较小。     

春玉米叶片衰老中激素变化,Ca^2＋跨膜运输和膜脂过氧化三者之间的关系

通过离体玉米（ZeamaysL．）叶片培养和叶肉质膜微囊45Ca2 吸收等实验,探索春玉米叶片衰老过程中激素变化、Ca2 跨膜运输及膜脂过氧化三者之间的联系。结果认为,玉米叶片衰老的可能过程首先是内源激素含量变化,继而影响到Ca2 跨膜运输,进而导致膜脂过氧化,由此引起叶绿素和蛋白质降解。     

pH值对紫色光合细菌Rhodopseudomonas palustris外周捕光天线的影响

采用稳态吸收光谱、圆二色谱、亚微秒时间分辨光谱手段检测了不同pH值对Rhodopseudomonas palustris的外周捕光天线LH2复合物的结构及色素功能的影响, 发现: (i) 在强酸性pH诱导下, B800细菌叶绿素分子在分钟时间尺度上逐渐转化成游离色素, 而类胡萝卜分子(Car)的光谱变化与B850的变化基本同步. 伴随着B800的缺失, 其Qy带对应的圆二色信号消失; (ii) 在强碱性条件下, B800分子比较稳定, B850分子的Qy带从852 nm蓝移至837 nm左右, 其近红外区域的CD 信号也发生了相应蓝移; 不同pH值条件下可见光区域Car分子均呈现出特征的CD信号; (iii) 在生理及碱性情况下, 采用532 nm激光脉冲直接激发Car分子, 在亚微秒时间尺度上观察到类胡萝卜素的TnT1吸收; 而在强酸性pH下仅观察到无特征的弱吸收带. 以上实验结果表明Rps. palustris的LH2复合物在酸性条件下B800发生缺失, Car的光保护功能受到影响; 而在强碱性条件下环状聚集体结构仍然保持, Car能正常发挥其光保护功能.     

CeNCl-CeO2 Heterojunction-Modified Ni Catalysts for Efficient Electroreduction of CO2 to CO

Renewable-electricity-powered electrochemical CO₂ reduction (CO₂RR) is considered one of the most promising ways to convert exhaust CO₂ into value-added chemicals and fuels. Among various CO₂RR products, CO is of great significance since it can be directly used as feedstock to produce chemical products through the Fischer–Tropsch process. However, the CO₂-to-CO electrocatalytic process is often accompanied by a kinetically competing side reaction: H₂ evolution reaction (HER). Designing electrocatalysts with tunable electronic structures is an attractive strategy to enhance CO selectivity. In this work, a CeNCl-CeO₂ heterojunction-modified Ni catalyst is successfully synthesized with high CO₂RR catalytic performance by the impregnation-calcination method. Benefiting from the strong electron interaction between the CeNCl-CeO₂ heterojunction and Ni nanoparticles (NPs), the catalytic performance is greatly improved. Maximal CO Faradaic efficiency (FE) is up to 90% at −0.8 V (vs RHE), plus good stability close to 12 h. Detailed electrochemical tests and density functional theory (DFT) calculation results reveal that the introduction of the CeNCl-CeO₂ heterojunction tunes the electronic structure of Ni NPs. The positively charged Ni center leads to an enhanced local electronic structure, thus promoting the activation of CO₂ and the adsorption of ^*COOH.     

Synthesis of novel NBD-GM1 and NBD-GM2 for the transfer activity of GM2-activator protein by a FRET-based assay system

The ganglioside-activator protein is an essential cofactor for the lysosomal degradation of ganglioside GM2 (GM2) by beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interphase. Mutations in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. In order to efficiently and sensitively probe the glycolipid binding and membrane activity of this cofactor, we synthesized two new fluorescent glycosphingolipid (GSL) probes, 2-NBD-GM1 and 2-NBD-GM2. Both compounds were synthesized in a convergent and multistep synthesis starting from the respective gangliosides isolated from natural sources. The added functionality of 2-aminogangliosides allowed us to introduce the chromophore into the region between the polar head group and the hydrophobic anchor of the lipid. Both fluorescent glycolipids exhibited an extremely low off-rate in model membranes and displayed very efficient resonance energy transfer to rhodamine-dioleoyl phosphoglycerol ethanolamine (rhodamine-PE) as acceptor. The binding to GM2-activator protein (GM2AP) and the degrading enzyme was shown to be unaltered compared to their natural analogues. A novel fluorescence-resonance energy transfer (FRET) assay was developed to monitor in real time the protein-mediated intervesicular transfer of these lipids from donor to acceptor liposomes. The data obtained indicate that this rapid and robust system presented here should serve as a valuable tool to probe quantitatively and comprehensively the membrane activity of GM2AP and other sphingolipid activator proteins and facilitate further structure-function studies aimed at delineating independently the lipid- and the enzyme-binding mode of these essential cofactors.     

Amplification of blue emission of Tm3+ ions in Li2O-HfO2-SiO2 glass system by means of Au0 metallic particles

Li₂O-HfO₂-SiO₂-Tm₂O₃:Au₂O₃ glass samples (containing fixed content of Tm₂O₃ and different concentration of Au₂O₃) were prepared and characterized. Bearing of Au⁰ metallic particles (MPs) on improving blue emission of thulium ions (Tm³⁺) ions was explored. Optical absorption (OA) spectra exhibited multiple bands excited from ³H₆ of Tm³⁺. Additionally, a broad peak in the wavelength range 500–600 nm due to surface plasmon resonance (SPR) of Au⁰ MPs was noticed in the spectra. Photoluminescence (PL) spectra (of thulium free glasses) indicated a peak in the visible range due to sp → d electronic transition of Au⁰ MPs. Luminescence spectra of Tm³⁺ and Au₂O₃ co-doped glasses exhibited intense blue emission with substantial increase of intensity with increase of Au₂O₃ content. Bearing of Au⁰ MPs on the reinforcement of blue emission of Tm³⁺ was discussed in detail with kinetic rate equations.     

2-Methylbutyryl CoA dehydrogenase from mitochondria of Ascarissuum and its relationship to NADH-dependent 2-methylcrotonyl CoA reduction

Acyl CoA dehydrogenase and electron-transfer flavoprotein have been isolated and partially purified from mitochondria of the anaerobic nematode, $\text{Ascaris}\text{suum}$. Dehydrogenase activity was greatest with 2-methylbutyryl CoA and the relative substrate specificities of the ascarid dehydrogenase(s) differ greatly from their mammalian counterparts. It appears that the ascarid dehydrogenase functions physiologically as a reductase, catalyzing the final step in the synthesis of branched-chain fatty acids. In fact, incubations of $\text{A}\text{.}\text{suum}$ mitochondrial membranes with electron-transfer flavoprotein, 2-methylbutyryl CoA dehydrogenase, 2-methylcrotonyl CoA and NADH resulted in a substantial, rotenone-sensitive, 2-methylbutyrate synthesis. These results suggest that the ascarid electron-transport chain and at least two soluble mitochondrial proteins are involved in the NADH-dependent reduction of 2-methylcrotonyl CoA.     

General O-glycosylation of 2-furfuryl alcohol using beta-glucuronidase

beta-Glucuronidase from bovine liver is able to catalyze transfer of several carbohydrates to furfuryl alcohol, an acid-sensitive diene, with transfer yields as high as 84%. Carbohydrates that were transferred in yields of 30% or higher include gluco-, galacto-, xylo-, and fucopyranose. Small variations in the configuration of the substrate hydroxyls lead to large variations in the catalytic behavior of the enzyme in terms of both the initial reaction velocities and the final ratios of transfer-to-hydrolysis. The high transfer yields and surprising nonspecificity towards carbohydrate suggest that the enzyme may be a versatile tool for the general O-glycosylation of dienic alcohols.     

Crystal structure of putidaredoxin, the [2Fe-2S] component of the P450cam monooxygenase system from Pseudomonas putida

Stability of the [2Fe-2S]-containing putidaredoxin (Pdx), the electron donor to cytochrome P450cam in Pseudomonas putida, was improved by mutating non-ligating cysteine residues, Cys73 and Cys85, to serine singly and in combination. The increasing order of stability is Cys73Ser/Cys85Ser>Cys73Ser>Cys85Ser>WT Pdx. Crystal structures of Cys73Ser/Cys85Ser and Cys73Ser mutants of Pdx, solved by single-wavelength anomalous dispersion phasing using the [2Fe-2S] iron atoms to 1.47 A and 1.65 A resolution, respectively, are nearly identical and very similar to those of bovine adrenodoxin (Adx) and Escherichia coli ferredoxin. However, unlike the Adx structure, no motion between the core and interaction domains of Pdx is observed. This higher conformational stability of Pdx might be due to the presence of a more extensive hydrogen bonding network at the interface between the two structural domains around the conserved His49. In particular, formation of a hydrogen bond between the side-chain of Tyr51 and the carbonyl oxygen atom of Glu77 and the presence of two well-ordered water molecules linking the interaction domain and the C-terminal peptide to the core of the molecule are unique to Pdx. The folding topology of the NMR model is similar to that of the X-ray structure of Pdx. The overall rmsd of Calpha positions between the two models is 1.59 A. The largest positional differences are observed for residues 18-21 and 33-37 in the loop regions and the C terminus. The latter two peptides display conformational heterogeneity in the crystal structures. Owing to flexibility, the aromatic ring of the C-terminal Trp106 can closely approach the side-chains of Asp38 and Thr47 (3.2-3.9 A) or move away and leave the active site solvent-exposed. Therefore, Trp106, previously shown to be important in the Pdr-to-Pdx and Pdx-to-P450cam electron transfer reactions is in a position to regulate and/or mediate electron transfer to or from the [2Fe-2S] center of Pdx.     

Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer

In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.     

糖脂转运蛋白家族新成员人类GLTPD2的生物信息学分析

人类糖脂转运结构域2蛋白(Glycolipid transfer protein domain containing 2,GLTPD2)是糖脂转运蛋白(Glycolipid trans-fer protein,GLTP)家族的一个新成员,其功能目前尚不清楚.研究的目的在于通过生物信息学分析,预测人类GLTPD2的结构、...     

Highly sensitive fluorescence detection of glycoprotein based on energy transfer between CuInS2 QDs and rhodamine B

A highly sensitive fluorescence method for glycoprotein detection has been established based on fluorescence resonance energy transfer (FRET) between CuInS₂ quantum dots (QDs) and rhodamine B (RB). Lectins comprise a group of proteins with unique affinities toward carbohydrate structures, so the process of FRET can occur between lectin‐coated QDs (CuInS₂ QDs–Con A conjugates, acceptors) and carbohydrate‐coated RB (RB–NH₂‐glu conjugates, donors). The fluorescence of lectin‐coated QDs was recovered in the presence of a glycoprotein such as glucose oxidase (GOx) and transferrin (TRF), which significantly reduced the FRET efficiency between the donor and the acceptor. Under optimal conditions, a linear correlation was established between the fluorescence intensity ratio I₆₅₄/I₅₇₇ and the TRF concentration over the range of 6.90 × 10^‐10 to 3.45 × 10^‐8 mol/L, with a detection limit of 2.5 × 10^‐10 mol/L. The linear range for GOx is 3.35 × 10^‐10 to 6.70 × 10^‐8 mol/L, with a detection limit of 1.5 × 10^‐10 mol/L. The proposed method was applied to the determination of glycoprotein in human serum and cell‐extract samples with satisfactory results. Furthermore, CuInS₂ QDs–Con A conjugates are used as safe and efficient optical nanoprobes in HepG2 cell imaging. Copyright © 2015 John Wiley & Sons, Ltd.