变藻蓝蛋白中能量传递过程的计算机模拟

藻胆体的变藻蓝蛋白 (allophycocyanin ,APC)核外联天线杆 ,内接光合反应中心 ,在能量传递中起着承上启下的作用 .根据X射线晶体结构数据和变藻蓝蛋白亚基的吸收和荧光光谱 ,以计算机模拟方法研究变藻蓝蛋白单体和三聚体中的能量传递过程 .计算结果表明 :变藻蓝蛋白单体中两个亚基之间能量传递性质与C 藻蓝蛋白 (C phycocyanin ,C PC)相似 ;而在三聚体时则与C 藻蓝蛋白三聚体有根本的区别 .无论什么聚集态 ,C 藻蓝蛋白中β84色团总是主要的荧光发射体 ;而在变藻蓝蛋白中α84则成为主要的荧光发射体 .这与C 藻蓝蛋白中能量传递的基本单元是六聚体而在变藻蓝蛋白中为三聚体的事实一致 .由此可以推断变藻蓝蛋白中 2个三聚体是以α84相接的面面接近方式 ,因而 2个三聚体间能量传递主要靠α84色团实现 .变藻蓝蛋白这种特殊性是保证其高效传能功能的关键     

螺旋藻变藻蓝蛋白发色团间相互作用机理研究

本文发现从螺旋藻(Spirulina platensis)中分离出的变藻蓝蛋白三聚体(αβ)_3在解聚为单体(αβ)时,伴随着明显的光谱学特性的变化。我们利用稳态和皮秒(10~(12)S)瞬态光谱学的方法研究了导致变藻蓝蛋白单体(αβ)和三聚体(αβ)_3光谱学差别的发色团之间的相互作用。计算了变藻蓝蛋白中发色团的吸收和发射跃迁偶极矩之间的夹角。尝试性地建立了用以解释变藻蓝蛋白内发色团之间的相互作用的激子相互作用和弱偶极相互作用的机理模型。     

多变鱼腥藻藻胆蛋白共价复合物的合成及其分子内能量传递

利用双功能基偶联剂3-(2-吡啶联巯基)丙酸N-羟基琥珀酰亚胺酯(SPDP)合成了两个藻胆蛋白复合物,藻红蓝蛋白-变藻蓝蛋白复合物PEC-APC和藻红蓝蛋白-藻蓝蛋白复合物PEC-PC.利用吸收光谱和荧光光谱证明了藻胆蛋白构型与构象在反应后得到保持。通过荧光光谱观察到能量传递现象。计算出复合物PEC-APC的分子内能量传递效率约为90%.复合物PEC-PC中藻红蓝蛋白PEC的荧光寿命比PEC本身的寿命大大缩短,证明存在分子内能量传递。二硫苏糖醇(DTT)还原二硫桥键后能量传递被阻断。这进一步证明复合物合成成功及分子内能量传递。     

C-藻蓝蛋白复合物能量传递过程动力学

藻类天线系统是多种藻胆蛋白和连接蛋白巧妙构成的有机功能体 .目前藻类天线系统中多种藻胆蛋白高分辨晶体结构已获得测定 ,但这些结构只是来源于孤立态的藻胆蛋白 ,因而 ,藻胆蛋白复合物的研究是了解不同藻胆蛋白之间联系的有效途径 .鉴于目前实验方法的困难 ,利用计算机模拟方法研究了C 藻蓝蛋白复合物内能量传递过程 .计算结果显示出复合物内能量传递的主要途径和动态性质 ;同时通过系统内 2个C 藻蓝蛋白六聚体盘之间激发能传递随时间的分布 ,发现快速的激发能盘间传递过程 .根据时间分辨光谱对激发能传递时间的定义 ,当以零时刻理想δ脉冲光激发第 1个盘中的色团时 ,第 2个盘内激发能上升时间仅几个皮秒 ;第 3盘的激发能上升时间小于 2 0ps .这些结果第一次直观显示出盘间传递的快速过程 ,为完整藻类天线内进行的快速、高效的能量传递提供了合理的解释 .     

藻胆体核心复合物结构与功能的研究（Ⅱ）

从螺旋藻藻胆体中分离出４种不同种结构和光谱形式的变藻蓝蛋白复合物ＡＰＩ、ＡＰⅡ、ＡＰⅢ和ＡＰＢ，利用吸收光谱、荧光光谱比较了三聚体和单体的光谱特性，通过对吸收光谱的光谱解叠以及各组分的归属，研究了变藻蓝蛋白复合物内各色团间相互作用的性质和在能量传递中的功能，结果表明，复合物内色团间的作用关系可以用Ｆｏｒｓｔｅｒ偶极－偶极作用机制来解释，由于妆蛋白和同源亚基的存在影响其结构的对称性，进而影响各色团间     

过渡金属离子(Cu2+、Cd2+)对藻红蛋白单体的漂白机制

藻胆蛋白是一组同源捕光色素蛋白,由多肽链及相连的开链四吡咯环色团构成。多肽链对色团的调控形成不同藻胆蛋白的特征吸收及荧光光谱。这些色团是藻蓝胆素(吸收带590～670 nm)、藻红胆素(吸收带530～570 nm)和藻尿胆素(吸收带490～500nm)。藻尿胆素由于在共轭体系中含有较少的双键,所以与前二者比,在较短波长有吸收。它们总是与藻红胆素伴生,在藻胆蛋白中作为敏化基团存在。     

藻胆体核心复合物结构与功能的研究(Ⅱ)──4种变藻蓝蛋白复合物发色团相互作用机制

从螺旋藻藻胆体中分离出4种不同结构和光谱形式的变藻蓝蛋白复合物APⅠ、APⅡ、APⅢ和APB, 利用吸收光谱、荧光光谱比较了三聚体和单体的光谱特性, 通过对吸收光谱的光谱解曾以及各组分的归属, 研究了变藻蓝蛋白复合物内各色团间相互作用的性质和在能量传递中的功能.结果表明, 复合物内色团间的作用关系可以用Forster偶极-隅极作用机制来解释, 由于连接蛋白和同源亚基的存在影响其结构的对称性, 进而影响各色团间相互作用的形式和性质.     

阴离子交换和羟基磷灰石色谱从螺旋藻中提纯藻胆蛋白的研究

本文采用离子交换层析和羟基磷灰石吸附层析技术从螺旋藻中提取纯化得到藻蓝蛋白和别藻蓝蛋白。通过比较 ＡＮＸ Ｓｅｐｈａｒｏｓｅ ４ Ｆａｓｔ Ｆｌｏｗ（ｈｉｇｈ ｓｕｂ／ｌｏｗ ｓｕｂ）、 ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ和 Ｑ Ｓａｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ等阴离子交换树脂的动态吸附容量以及目标产品的纯度,选用 ＤＥＡＥＳｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ作为层析介质．对离子交换的产物进行了电泳分析,藻蓝蛋白和别藻蓝蛋白的等电点接近,电迁移速率相似。采用羟基磷灰石吸附技术对藻胆蛋白混合物进一步分离纯化,分别得到了藻蓝蛋白和别藻蓝蛋白的纯品,经等电聚焦实验验证显示为均一组成。     

阴离子交换和羟基磷灰石色谱从螺旋灌中提纯藻胆蛋白的研究

本文采用离子交换层析和羟基磷灰石吸附层析技术从螺旋灌中提取纯化得到藻蓝蛋白和别藻蓝蛋白。通过比较ANX Sepharose 4Fast Flow(high sub/low sub)、DEAE Sepharose Fast Flow和Q Sepharose Fast Flow等阴离子交换树脂的动态吸附容量以及目标产品的纯度,选用DEAE Sepharose Fast Flow作为层析介质。对离子交换的产物进行了电泳分析,藻蓝蛋白和别藻蓝蛋白的等电点接近,电迁移速率相似。采用羟基磷灰石吸附技术对藻胆蛋白混合物进一步分离纯化,分别得到了藻蓝蛋白和别藻蓝蛋白的纯品,经等电聚焦实验验证显示为均一组成。     

藻胆色素与半胱氨酸残基的结合

本文对藻胆色素与半胱氨酸残基的结合反应进行了试验,通过藻红胆素二甲酯、藻蓝胆素二甲酯分别与半胱氨酸甲酯、谷胱甘肽(还原型)二甲酯的反应得到了一些与藻胆蛋白上发色团具有相同共价结构的化合物。这些化合物与其他胆色素类化合物不同的是有很强的荧光发射和圆二色效应。化合物的圆二色效应分析表明,藻胆色素与半胱氨酸甲酯、谷胱甘肽(还原型)二甲酯反应时得到的产量较大的异构体与藻胆蛋白上的发色团具有相同的立体结构。     

Computer simulation on the energy transfer processes in allophycocyanins

Ｐｈｙｃｏｂｉｌｉｐｒｏｔｅｉｎｓａｒｅｔｈｅｌｉｇｈｔｈａｒｖｅｓｔｉｎｇｐｉｇｍｅｎｔｓｆｏｒｐｈｏｔｏｓｙｎｔｈｅｓｉｓｉｎａｌｇａｅａｎｄｉｎｃｌｕｄｅｐｈｙｃｏｅｒｙｔｈｒｉｎ,ｐｈｙｃｏｃｙａｎｉｎａｎｄａｌｌｏｐｈｙｃｏｃｙａｎｉｎ.Ｉｎｒｅｄａｎｄｂｌｕｅａｌｇａｅ,ｄｉｆｆｅｒｅｎｔｋｉｎｄｓｏｆｐｈｙｃｏｂｉｌｉｐｒｏｔｅｉｎｓａｓｗｅｌｌａｓｌｉｎｋｅｒｐｏｌｙｐｅｐｔｉｄｅｓｆｏｒｍａｗｅｌｌｏｒｇｎｉｚｅｄｓｙｓｔｅｍｗｉｔｈｅｆｆｉｃｉｅｎｔｆｕｎｃｔｉｏｎｓｏｆｌｉｇ…     

ENERGY TRANSFER IN TRIMERIC C-PHYCOCYANIN STUDIED BY PICOSECOND FLUORESCENCE KINETICS

Abstract— The excited state kinetics of trimeric C-phycocyanin from Mastigocladus laminosus has been measured as a function of the emission and excitation wavelength by the single-photon timing technique with picosecond resolution and simultaneous data analysis. A fast decay component of 22 ps (C-phycocyanin with linker peptides) and 36 ps (C-phycocyanin lacking linker peptides) is attributed to efficient energy transfer from sensitizing to fluorescing chromophores. At long detection wavelengths the fast decay components are found to turn into a rise term. This finding further corroborates the concept of intramolecular energy transfer. Previous reports on the conformational heterogeneity of the chromophores and/or proteins in C-phycocyanin are confirmed. Our data also provide indications for the importance of the uncoloured linker peptides for this heterogeneity.     

PHOTOREACTIONS OF THE CHROMOPHORE CATIONS OF DENATURED SPECIES OF C-PHYCOCYANIN AND ALLOPHYCOCYANIN

Abstract— C-phycocyanin dissolved in buffer containing 75% ethylene glycol (vol/vol) shows photorevers-ible reactions which are ascribed to the interconversion by light of two species: PC_r, PC_g. After denaturation with formic acid, the chromophore cation of PC_g, can be irreversibly photoconverted to that of PC_r A conversion of the chromophore of denatured PC_g, to that of denatured PC_r also occurs after adjustment of the pH to about 8. Under the same conditions the chromophore of allophycocyanin shows similar reactions. Moreover, the reactions of the chromophores of the denatured species of these phycobiliproteins are similar to the reactions of the chromophore of the denatured species P_fr of photochrome.     

The Use of Carrier Ampholyte-Free Isoelectric Focusing for Proteomic Analysis

Carrier ampholyte-free isoelectric focusing was applied for pre-concentration, purification and micropreparation of phycobiliproteins (C-phycocyanin, allophycocyanin, B-phycoerythrin) extracted from cyanobacteria Anabeana doliolum and from red microalga Porphyridium cruentum. The extraction of phycobiliproteins was carried out in deionized water. The sonication in the ultrasonic bath and liquid nitrogen freeze grind was used for extraction of proteins of interest. Pre-concentrated and pre-separated proteins were collected and analyzed via MALDI-TOF-TOF mass spectrometer after their proteolytic digestion via trypsin. Based on tandem mass spectrometric analysis, the C-phycocyanin, allophycocyanin and B-phycoerythrin were identified unambiguously.

     

The Use of Carrier Ampholyte-Free Isoelectric Focusing for Proteomic Analysis

Carrier ampholyte-free isoelectric focusing was applied for pre-concentration, purification and micropreparation of phycobiliproteins (C-phycocyanin, allophycocyanin, B-phycoerythrin) extracted from cyanobacteria Anabeana doliolum and from red microalga Porphyridium cruentum. The extraction of phycobiliproteins was carried out in deionized water. The sonication in the ultrasonic bath and liquid nitrogen freeze grind was used for extraction of proteins of interest. Pre-concentrated and pre-separated proteins were collected and analyzed via MALDI-TOF-TOF mass spectrometer after their proteolytic digestion via trypsin. Based on tandem mass spectrometric analysis, the C-phycocyanin, allophycocyanin and B-phycoerythrin were identified unambiguously.     

STRUCTURAL DIFFERENCES BETWEEN PHYCOCYANIN and ALLOPHYCOCYANIN FROM THEIR RESONANCE RAMAN SPECTRA*

Abstract— Resonance Raman spectra of the chromophores of the cyanobacterial light-harvesting proteins phycocyanin (CPC) and allophycocyanin (APC) were recorded using 364 nm excitation. The1500–1700 cm^-1 regions of these spectra were analyzed for the pH-induced structural changes accompanying the disruption of the native trimers into monomers as well as the progressive denaturation of these monomers. Computer-assisted decomposition of the 1642 cm^-1 marker bands of these spectra yielded up to four components (named I-IV), the frequencies of which were constant within 5 cm^-1 (CPC) and 10 cm^-1 (APC). The relative intensities of two of these components, namely I and III, were sensitive to chromophore conformations. The previously reported downshift of the 1642 cm^-1 band upon folding of the chromophores was shown to result from a weakening of component I and a concomitant enhancement of component III. Components I-IV had different relative intensity patterns in CPC and APC spectra. In particular, the higher relative intensity of component I at 1646 cm^-1 indicated more extended average conformations of the chromophores in trimeric APC than in trimeric CPC. This difference likely resulted from the extra β-155 chromophore present in CPC. Component III was sizably active in RR spectra of monomelic APC but was not observed either in those of monomelic CPC or in those of trimeric APC and CPC. This indicated that, in APC monomers, chromophore(s) did not assume the native conformations found in the trimer, while monomer formation did not sizably alter the structures of the CPC chromophores.     

Kinetics of energy transfer processes in C-phycocyanin complexes

The antenna system of algae for photosynthesis is a functional entity composed of various phycobiliproteins and the linker polypeptides. Up to now, high-resolution crystal structure data have been available only for the isolated phycobiliproteins. To have an understanding of the functional connection between different phycobiliproteins, it is necessary to study the complexes composed of different phycobiliproteins. The energy transfer processes in C-phycocyanin complexes were studied through computer simulation because it is difficult to be studied by conventional experimental methods. The main pathways of energy flow and the dynamic property of the energy transfer were obtained. A fast transfer process between two neighboring disks was observed through analyzing the distribution curves of excitation energy over time. According to the definition of the time constants for energy transfer in time-resolved spectrum techniques, for a complex with three C-phycoeyanin hexamer disks, a fluorescence-rising comp     

FÖRSTER TRANSFER CALCULATIONS BASED ON CRYSTAL STRUCTURE DATA FROM Agmenellum quadruplicatum C-PHYCOCYANIN

Excitation energy transfer in C-phycocyanin is modeled using the Forster inductive resonance mechanism. Detailed calculations are carried out using coordinates and orientations of the chromophores derived from X-ray crystallographic studies of C-phycocyanin from two different species (Schirmer et al, J. Mol. Biol. 184 , 257–277 (1985) and ibid. , 188 , 651-677 (1986)). Spectral overlap integrals are estimated from absorption and fluorescence spectra of C-phycocyanin of Mastigocladus laminosus and its separated subunits. Calculations are carried out for the β-subunit, αβ-monomer, (αβ)₃-trimer and (αβ)₀-hexamer species with the following chromophore assignments: β155 = 's'(sensitizer), β84 ='f (fluorescer) and α84 ='m'(intermediate):]:. The calculations show that excitation transfer relaxation occurs to 3=98% within 200 ps in nearly every case; however, the rates increase as much as 10-fold for the higher aggregates. Comparison with experimental data on fluorescence decay and depolarization kinetics from the literature shows qualitative agreement with these calculations. We conclude that Forster transfer is sufficient to account for all of the observed fluorescence properties of C-phycocyanin in aggregation states up to the hexamer and in the absence of linker polypeptides.     

Expanded Bed Adsorption Chromatography for Recovery of Phycocyanins from the Microalga Spirulina Platensis

We carried out the purification of C-phycocyanin and allophycocyanin from Spirulina platensis taking advantage of the adsorption properties of the expanded beds. Initially, phycobiliproteins were released from the microalga cells by osmotic shock. Next, phycocyanins were recovered by applying the centrifuged cell suspension directly to the anion exchanger Streamline-DEAE using expanded bed columns, equilibrated with 50 mM sodium phosphate buffer, pH 7.0. After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and phycocyanins rich solution was eluted with a downward flow of 500 mM sodium phosphate buffer, pH 7.0. Finally, we utilized conventional gel filtration and ion exchange chromatography methods for separation and purification of C-phycocyanin and allophycocyanin. The purification steps were monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the purity of recovered phycocyanins was confirmed by absorption and emission spectroscopy. The main advantage of this new method is the high yield achieved in the steps of product extraction and adsorption by expanded bed adsorption, so reducing both processing times and costs.