Diversity of pigment-protein complexes of photosynthetic organisms.

PS I, PS II and light-harvesting complexes (LHC) in oxygen evolving photosynthetic organisms were reviewed. These organisms include cyanobacteria, red algae, brown algae, diatoms, chrysophytes, dinophytes, xanthophytes, crypophytes, green algae and green plants. The diversity of pigment-protein complexes that fuel the conversion of radiant energy to chemical bond energy was highlighted, and the evolutionary relationships among the LHC structural polypeptides and the characteristics of the fluorescence emission of PS I at 77 K was discussed.

Isolation and nomenclature of pigment-protein complexes from the brown alga (Undaria pinnatifida Harv.)

Eight kinds of pigment-protein complexes were resolved from the thylakoid membrane of the brown alga (Undaria pinnatifida Harv.) by using non-ionic detergent decanoyl-N-methylglucamide and PAGE technique. According to the apparent molecular weights, spectra characteristics, polypeptide compositions and referring to the higher plant spinach, eight pigment-protein complexes were named under Anderson's terminology system as CP I a, CP I, CPa, LHC1, LHC2, LHC3, LHC4, LHC5.

Studies of non-covalent protein complexes by MALDI methods

Several specific non-covalent protein complexes were successfully observed by matrix assisted desorption ionization mass spectrometry(MALDI MS). The methods described in this paper include the matrixes use of sinapinic acid(SA) and 6-aza-2-thiothymine (ATT) in neutral pH solution, as well as the improvement of two-layer sample preparation method to achieve a high sensitivity detection of stable non-covalent complexes, Myoglobin-heme complex was found simultaneously with the sinapinic acid matrix in the various pH solution(pH=2 or pH=5), The RNase S complex showed a striking intensity at the first shot, which was decreased with more laser shots. Most importantly, the observation of specific non-covalent complex in the brome mosaic virus(BMV) coat proteins would open up a new possibility to investigate the assembly and disassembly of viral capsids.

Characteristics and comparative study of chlorophyll-protein complexes from siphonous green algae

By mild PAGE method, 11, 11, 7 and 9 chlorophyll-protein complexes were isolated from two species of siphonous green algae ( Codium fragile (Sur.) Harlot and Bryopsis corticulans Setch.), green alga (Ulothrix flacca (Dillw.) Thur.), and spinach (Spinacia oleracea Mill.), respectively. Apparent molecular weights, Chi a/b ratios, distribution of chlorophyll, absorption spectra, low temperature fluorescence spectra of these complexes were determined, and compared with one another. PS I complexes of two siphonous green algae are larger in apparent molecular weight because of the attachment of relative highly aggregated LHC I. Four isolated light-harvesting complexes of PSII are all siphonaxanthin-Chl a/b-protein complexes, and they are not monomers and oligomers like those in higher plants. Especially, the absence of 730 nn fluorescence in PS I complexes indicates a distinct structure and energy transfer pattern.

高等植物PSI ,PSII反应中心及天线系统色素蛋白复合体结构与功能的研究

一、实验证明了Cd H、Mg“在小麦类囊体膜上色素蛋白复合体的解聚和再聚合过程中，具有不同的作用。因此，二阶阳离子对激发能在光系统间的分配调节作用，可能不能仅仅用“静电现象”(Barber(1980))去解释。分析表明，在Ca2+作用下与PSII内周天线CP-47，GP-43多肽结合的L H C II和LHClb是来自间质膜区的PS I系统的。从PSI迁移到P S II的捕光色素蛋白，增加了PSII的捕光截面，从而促进了激发能有利于PsII分配。 二、Ca2*、Mg2+对小麦和菠菜类囊体膜光谱性质的影响有所差异。Ca2+对小麦类囊体膜光谱性质的影响还可以随着介质中Ca2+的消除而消除。同小麦类囊体膜相比，菠菜PSII以及LHCII更为集中在基粒区域，这可能是菠菜类囊体膜强Fv以及高F888／F735，F89H／F735比值的原因。因此，Ca2+，HgH对激发能在光系统间分配的调节作用是依赖于光系统间激发能及天线色素蛋白的分配状况的。 三、对菠菜叶中分离的PSII-RC: D1-D2-cyt b55g复合物进行的低温荧光发射光谱的研究表明，这一复合物可能具有F681和F684两种波长的低温荧光发射，但它们通常并不是同时存在，而是取决于Ca-670与Ca-680 Chla分子的相对含量的。PSII-RC内周无线GP-47，GP-43多肽的存在是D1-D2-cyt b559复合物低温荧光发射红移的原因;而D1一D2cyt b559复合物的不稳定性则与其低温荧光发射的蓝移现象有关。 从蕹菜叶中分离的Dl—D2-cyt b559复合物的F 381低温荧光发射也是由其相对含量较高的C．i-6 7 0 Chla分子的存在决定的。对蕹菜D 1一D 2-cyt b559复合物中的分析还表明，F 681的低温荧光发射直接来源于Di／D2复合物，而415nm处相对较强的吸收，则可能主要是与Pheo的存在有关的。 四、多肽分析与光谱分析的对照表明，CP-26内周天线多肽可能是PSII中F695低温荧光发射的真正来源。 五、实验分析了蔗糖密度离心分离的LHClI和PSI颗粒。结果排除了CP-27多肽（以及CP，一2 5，GP-47，CP -4 3多肽）具有F695低温荧光发射的可能，因此支持了CP-26多肽是PSII中F695低荧光发射来源的看法。对PsI颗粒的分析表明，P700的存在可能是与PSI-RC中较大的Sub-I亚基相联系的。 六、根据以上的研究结果，提出了PSI，PSII在类囊体膜上的结构模式，并对其内容进行了分析和讨论。

PSII色素蛋白复合物的脂质体重组及反应中心的功能研究

光系统II（PSII）是存在于类囊体膜中的多亚基色素蛋白复合物，是吸收光能、催化光诱导水裂解释放氧气、质子和电子的重要机构。它在体内的基本单位是由外周天线蛋白（LHCII）与PSII核心复合物结合形成的PSII-LHCII超分子复合物，这一结构保证了LHCII吸收的能量能够快速有效的传递到PSII反应中心（RC），进行原初光化学反应。 本论文分为两部分：1、利用捕光色素蛋白复合物（LHCII）与PSII核心复合物在以DGDG、PG、SQDG三种类囊体膜脂形成的脂质体中重组的方法，研究了LHCII与PSII在脂膜上结构与功能的相互作用;2、通过研究光破坏和色素置换对PSII RC的影响，探讨了RC中不同色素的功能。主要结果如下： 1、LHCII与PSII核心复合物的蛋白脂质体研究： 将OECC（粗提核心复合物）、pdOE（纯化核心复合物）、LHCII（大量天线）制剂分别与脂质体重组并研究了其光谱性质。LHCII在与脂质体重组前表现出典型的聚集态光谱特征，重组后吸收和荧光发射峰发生明显蓝移;LHCII、OECC和pdOE三种蛋白脂质体与重组前的样品相比荧光发射强度增加;表明脂环境影响了色素蛋白复合物的聚集状态以及色素和蛋白之间的相互作用。 OECC和pdOE分别与LHCII在脂质体中重组，得到两种重组蛋白（LHCII-OECC和LHCII-pdOE）脂质体，用冰冻蚀刻电镜技术和低温荧光光谱的方法研究其结构和功能特征。LHCII和核心复合物（OECC或pdOE）结合形成PSII-LHCII重组颗粒，并在脂质体中均匀排布，阻止了LHCII晶格状结构的形成。重组蛋白脂质体的吸收光谱既有LHCII的吸收特征，又有核心复合物的特征吸收峰，但低温荧光光谱的主要发射峰是核心复合物的特征峰（684 nm-685 nm），而不是LHCII的特征峰（680 nm）;而且激发不同色素得到的荧光发射光谱基本一致，这些结果证明LHCII吸收的能量传递到了核心复合物中，在重组蛋白脂质体中不同色素蛋白复合物在结构和功能上都实现了相互偶联。 通过对OECC或pdOE与LHCII重组形成的蛋白脂质体放氧或DCPIP光还原活性的检测研究了PSII光化学活性特征。LHCII和核心复合物（OECC或pdOE）的重组蛋白脂质体与单独核心脂质体相比，在强光和弱光下光化学活性都明显提高。这从另一个角度证明了核心复合物与LHCII的功能偶联，LHCII的结合使捕光截面积增大，从而使PSII光化学活性增加。 用77K飞秒时间分辨荧光光谱分析了几种蛋白脂质体的能量传递和捕获情况。LHCII、OECC和pdOE三种蛋白脂质体的主要荧光衰减组分分别是670 ps（发射峰在680 nm）、650 ps（发射峰在690 nm）和570 ps（发射峰在685 nm）。LHCII-OECC和LHCII-pdOE脂质体的主要衰减组分分别是940 ps（发射峰在690 nm）和840 ps（发射峰在685 nm），并且出现了一个在核心复合物脂质体和LHCII脂质体中没有的40 ps组分，可以推测，这是LHCII和核心复合物之间达到平衡的时间组分，比激发态衰减的平均寿命要快得多，因此支持了PSII的trap-limited激发能衰减动力学模型。此外，可以看到天线的增大使Chl a荧光衰减的寿命延长，这一特性可能与PSII的光保护机制有关。 LHCII和OECC、LHCII和pdOE在脂质体中都成功的实现了重组，而且在结构和功能上没有明显差异;表明小天线以及23 kDa、17 kDa蛋白可能不是LHCII和核心复合物结合及能量传递所必需的。 2、受体侧光破坏和色素置换对PSII RC的影响： 在800 μmol.m-2 .s-1光照和无外加电子受体、供体的情况下，研究了PSII RC色素的受体侧光破坏情况。Chl a、Pheo和β-Car的光漂白几乎同时发生，其中在680 nm吸收的色素破坏最为显著，670 nm吸收的外周Chl比其他色素更加稳定。荧光发射强度呈先升高后降低的趋势，最大发射峰位逐渐蓝移，表明色素之间的能量传递受到破坏。用β-Car的主要吸收波长488 nm和514.5 nm激发得到两组谱带峰位和强度不同的拉曼光谱，表明在PSII RC中存在两个光谱性质不同的β-Car。光破坏过程中两组谱带的位置和带宽都没有明显变化，表明β-Car的光保护机制不涉及自身构象的变化。 将PSII RC与Cu-Chl a进行色素置换，得到了与Cu-Chl重组的RC（Cu-Chl-RC），含有0.5 Cu-Chl/2Pheo。与对照RC（按同样方式与Chl a置换的RC）和天然RC相比，Cu-Chl含量增加而Chl含量减少，660 nm的吸收增加而670 nm吸收降低，因此推测是外周Chl被替换。色素置换过程对RC的多肽组分及大部分的P680活性没有影响，CD光谱的变化也很小，表明产生CD信号的色素和蛋白环境也没有受到明显影响。但是Cu-Chl-RC的荧光发射强度明显降低，最大发射峰蓝移且峰形发生变化，Cu-Chl可能在重组RC中作为激发态的淬灭剂，阻碍了色素之间的能量传递。

Consensus scoring for enriching near-native structures from protein-protein docking decoys

The identification of near native protein-protein complexes among a set of decoys remains highly challenging. A stategy for improving the success rate of near native detection is to enrich near native docking decoys in a small number of top ranked decoys. Recently, we found that a combination of three scoring functions (energy, conservation, and interface propensity) can predict the location of binding interface regions with reasonable accuracy. Here, these three scoring functions are modified and combined into a consensus scoring function called ENDES for enriching near native docking decoys. We found that all individual scores result in enrichment for the majority of 28 targets in ZDOCK2.3 decoy set and the 22 targets in Benchmark 2.0. Among the three scores, the interface propensity score yields the highest enrichment in both sets of protein complexes. When these scores are combined into the ENDES consensus score, a significant increase in enrichment of near-native structures is found. For example, when 2000 dock decoys are reduced to 200 decoys by ENDES, the fraction of near-native structures in docking decoys increases by a factor of about six in average. ENDES was implemented into a computer program that is available for download at http://sparks.informatics.iupui.edu.

Molecular Dynamics Simulation of Barnacle Cement

Barnacle cement is an underwater adhesive that is used for permanent settlement. Its main components are insoluble protein complexes that have not been fully studied. In present article, we chose two proteins of barnacle cement for study, 36-KD protein and Mrcp-100K protein. In order to investigate the characteristic of above two proteins, we introduced the method of molecular modeling. And the simulation package GROMACS was used to simulate the behavior of these proteins. In this article, before the simulations, we introduce some theories to predict the time scale for polymer relaxation. During the simulation, we mainly focus on two properties of these two proteins: structural stability and adhesive force to substrate. First, we simulate the structural stability of two proteins in water, and then the stability of 36-KD protein in seawater environment is investigated.We find that the stability varies in the different environments. Next, to study adhesive ability of two proteins, we simulate the process of peeling the two proteins from the substrate (graphite). Then, we analyze the main reasons of these results. We find that hydrogen bonds in proteins play an important role in the protein stability. In the process of the peeling, we use Lennard–Jones 12-6 potential to calculate the van der Waals interactions between proteins and substrate.

Molecular Dynamics Simulation of Barnacle Cement

Barnacle cement is an underwater adhesive that is used for permanent settlement. Its main components are insoluble protein complexes that have not been fully studied. In present article, we chose two proteins of barnacle cement for study, 36-KD protein and Mrcp-100K protein. In order to investigate the characteristic of above two proteins, we introduced the method of molecular modeling. And the simulation package GROMACS was used to simulate the behavior of these proteins. In this article, before the simulations, we introduce some theories to predict the time scale for polymer relaxation. During the simulation, we mainly focus on two properties of these two proteins: structural stability and adhesive force to substrate. First, we simulate the structural stability of two proteins in water, and then the stability of 36-KD protein in seawater environment is investigated. We find that the stability varies in the different environments. Next, to study adhesive ability of two proteins, we simulate the process of peeling the two proteins from the substrate (graphite). Then, we analyze the main reasons of these results. We find that hydrogen bonds in proteins play an important role in the protein stability. In the process of the peeling, we use Lennard-Jones 12-6 potential to calculate the van der Waals interactions between proteins and substrate.

牛耳草Bose hygrometrica 离体叶脱水-复水过程的光合作用及基因表达

本文以复苏植物牛耳草Boea hygrometrica成熟植株的离体叶片为试材，对比非复苏植物烟叶唇苣苔Chirita heterotricha, 以光合作用在脱水－复水过程中的变化为切入点，从生理水平上探讨其脱水保护位点：应用mRNA差异显示技术，从分子水平上探讨其脱水保护机制。 光合放氧速率、快速荧光诱导动力学、慢速荧光诱导动力学、荧光发射光谱、荧光激发谱的结果表明，相对于烟叶唇柱苣苔，脱水对牛耳草净光合速率、PS II和PS I光化学活性、电子传递、光合磷酸化及CO_2固定的影响有一个共同的特点，即脱水时迅速降低，复水后恢复能力强。通过非变性绿胶的研究牛耳草叶片类囊体膜叶绿素－蛋白复合体在脱水－复水过程中保持高度稳定。色素含量分析表明牛耳草的叶绿素含量在脱水－复水过程中也相对稳定。这些特征可能是牛耳草叶片光合作用脱水保护机制的一部分。 SDS－PAGE和IEF电泳结果表明，牛耳草脱水复苏过程中蛋白质表达有差异，或增或减，并分别发现了一条（SDS－PAGE）和两条（IEF）在脱水过程中特异出现的蛋白质。 本文以银染法代替放射自显影用于mRNA差异显示，不但简化了实验步骤，缩短了实验周期，而且在不降低灵敏度的前提下避免了放射性危害，降低了实验成本。本文证明了mRNA差异银染显示法用于复苏植物牛耳草脱水－复水过程中基因表达变化的研究是可行的。 mRNA差异银染显示法揭示牛耳草耐脱水复苏机制涉及到基因表达的调控。脱水－复水过程中差异表达的基因有6种，其中脱水特异诱导表达的13个cDNA所相应的基因、脱水上调节的15个cDNA所相应的基因可能参与牛耳草叶片脱水保护机制，复水特异诱导的8个cDNA的所相应基因可能参与牛耳草复水后的修复机制。2个脱水特异诱导表达的cDNA片段进行了克隆和测序。

高等植物PSI颗粒的分离提纯及其光破坏机理的初步探讨

从菠菜叶绿体中分离提纯PSI颗粒及其捕光天线色素蛋白复合物LHCI，对其光谱特性进行分析。对PSI颗粒中色素和蛋白的光破坏进程，并对外加组氨酸、Triton，以及温度对PSI颗粒光破坏的影响等进行了比较系统的研究，以探讨PSI光破坏的机理。其主要结果如下： 1. 对PSI颗粒和LHCI色素蛋白复合物的荧光光谱的研究，发现PSI中Chlb所吸收的光能主要传递给LHCI中的“长波组分”（吸收波长大于P700的Chla）。 2. 在PSI颗粒光破坏进程的研究中发现，Chla中吸收波长较长的组分首先发生光破坏;位于PSI颗粒外围的LHCI上的Chlb，也容易受到光破坏;Car先于Chlb发生光破坏。在光照处理过程中，PSI的天线色素蛋白复合物LHCI多肽降解程度大于反应中心多肽组分（PsaA，PsaB）的降解，其中LHCI-680首先由于光破坏而发生降解。PsaD也是容易受到光破坏而发生降解的一个多肽。另外，还发现在长时间光照后有蛋白聚合现象发生。 3. 在PSI颗粒中外加单线态氧的淬灭剂组氨酸，分析不同光强光照处理过程中组氨酸对PSI颗粒中色素和多肽光破坏的保护作用，发现外加组氨酸对强光照（2300μEm-2s-1）引起的叶绿素光吸收减少和CD信号减弱的有效抑制表现出一个明显的延迟期，但对强光诱导的荧光产量下降的效应却能立即表现出来;在强光照前期和弱光照（300μEm-2s-1）条件下，组氨酸不能抑制PSI颗粒的光吸收下降。另外，外加组氨酸除了对反应中心多肽有光保护作用以外，对PSI中其它多肽也有显著的保护作用。 4. 用不同浓度的Triton处理PSI颗粒，发现较低浓度的Triton可以增大叶绿素的光吸收和PSI颗粒的荧光产量，而不对PSI颗粒的多肽组成造成影响;当Triton浓度达到一定的程度时，虽然不会影响PSI颗粒的多肽组成，但是会使其光吸收减少，荧光产量下降;而当Triton浓度过高时，PSI颗粒的多肽会发生降解现象，同时其光吸收和荧光产量也迅速下降。Triton浓度较低时，PSI颗粒光破坏的程度随Triton浓度的增大而增大，当Triton浓度增大到一定的程度时，PSI颗粒的光破坏程度同Triton浓度不再呈明显的正相关。 5. 对PSI颗粒进行不同温度的热处理，其结果表明：温度较低（20 ℃～40 ℃）的热处理对PSI颗粒的多肽和叶绿素光吸收的影响程度很小，照光后不同温度热处理过的PSI颗粒光吸收减少和多肽降解的程度相近;温度较高（50 ℃～60 ℃）的热处理会对PSI颗粒的结构产生影响，使之稳定性减小，对光处理更敏感;温度更高（大于70 ℃）的热处理会破坏PSI颗粒的结构，引起多肽组分的降解。另外，不同的多肽对热处理的敏感性显著不同。 6. 低温（4 ℃）和常温（20 ℃）下PSI颗粒光破坏的比较发现，室温下PSI颗粒的光破坏程度明显大于低温下光破坏的程度，表明光处理过程中温度会影响到PSI颗粒光破坏的程度。 通过上述的研究结果，分析了PSI颗粒光破坏过程中色素和蛋白的变化及其外界因子的影响，对PSI颗粒光破坏的机制进行了初步的探讨。

Atomic force microscopic study of low temperature induced disassembly of RecA-dsDNA filaments

The assembly and disassembly of RecA-DNA nucleoprotein filaments on double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) are important steps for homologous recombination and DNA repair. The assembly and disassembly of the nucleoprotein filaments are sensitive to the reaction conditions. In this work, we investigated different morphologies of the formed nucleoprotein filaments at low temperature under different solution conditions by atomic force microscopy (AFM). We found that low temperature and long keeping time could induce the incomplete disassembly of the formed nucleoprotein filaments.