用蛋白内源荧光法考察盐溶液中两种外周蛋白构象的变化

作者借助由278nm和295nm光源激发的蛋白质内源荧光分析，考察了在几种盐溶液(甲醇和NaCl，NaCl，脲，三氯乙酸，CaCl2)中23kD，17kD蛋白构象的变化。结果表明，由295nm波长的光激发时，17kD蛋白荧光发射峰位于328nm处和360nm附近，这表明大部分17kD蛋白的Trp^71处于分子内部；23kD蛋白具有326nm荧光发射峰，表明23kD蛋白所含2个色氨酸残基(Trp^34和Trp^168)处于其分子内部的疏水部位。CaCl2、脲、NaCl、三氯乙酸、甲醇和NaCl对17kD和23kD外周蛋白的内源荧光都有影响，其中CaCl2、甲醇和NaCl的影响较大，脲、NaCl的影响相对较小，这反映了两种蛋白在溶液中的构象易发生改变。     

用蛋白内源荧光考察溶液pH对PSⅡ两种外周蛋白构象的影响

借助278nm和295nm激发的内源荧光光谱分析，考察不同pH值缓冲液中23kD、17kD蛋白构象变化．结果表明：295nm波长光激发时，23kD蛋白具有326nm荧光发射峰和360nm副峰；17kD蛋白具有328nm荧光发射峰和355nm副峰；278nm波长光激发时，23kD和17kD蛋白的最大荧光发射峰均在306nm处．与中性条件下相比，酸性(pH3．0～4．0)或碱性(pH8．0～9．0)缓冲液中，23kD蛋白和17kD蛋白的内源荧光光谱都发生显著变化：最大荧光峰位置和强度均不相同，340nm-360nm荧光发射副峰的强度增加．反映溶液中两种蛋白的构象易发生改变，离子键和氢键是维系23kD、17kD蛋白构象的重要因素之一。     

荧光光谱法研究甲基蓝与羊毛角蛋白的相互作用

在0.1 mol/L Tris-HC1弱酸和中性缓冲液中,采用荧光光谱法研究了甲基蓝(MB)与羊毛a-角蛋白相互作用.结果表明:在25℃和37℃,pH分别为5.45、6.25和7.20,MB与a-角蛋白结合形成1:1配合物,猝灭蛋白质内源荧光,敏化MB峰位在460 nm处的荧光.它们之间的结合反应是吸热、熵增驱动的自发过程,即反应过程中△H＞0和△S＞0,则其主要作用力是疏水作用.中性pH时其结合常数增大,表明中性环境蛋白质的局部构象更有利于MB的疏水结合.同时,37℃与25℃结合常数相比,37℃环境的KA大于25℃,与反应过程是吸热过程即△H＞0的结果一致.     

微咸水灌溉对玉米生长特性和产量影响研究

通过灌溉含有NaCl和CaCl2的微咸水玉米微区试验,分析了微咸水中NaCl浓度和CaCl2浓度对玉米生长特性和产量的影响。研究表明:灌溉不同NaCl浓度和CaCl2浓度的微咸水下,玉米的株高和叶面积随着生长时间的延长而增大;在相同生长时间里,玉米的株高和叶面积随着微咸水中NaCl浓度的增高而逐渐减小,随着微咸水中CaCl2浓度的增高而逐渐增大,但影响规律没有NaCl明显。玉米单株产量随着微咸水中NaCl浓度的增高而逐渐减少,而随着微咸水中CaCl2浓度的增高而逐渐增加。以上研究可以为盐碱地农作物种植提供理论依据。     

Ca^2＋和Mg^2＋对54kD钙结合蛋白荧光强度影响的研究

猪血小板 5 4kD钙结合蛋白 (calciumbindingprotein ,CaBP)是一个等电点为 5 .5的弱酸性蛋白质 ,其N 末端为丙氨酸 (Ala) ,且未经修饰 ,在 2 80nm波长的紫外光激发下 ,该蛋白质具有λmax为 335nm的内源荧光发射光谱 ,Ca2 +、Mg2 +均能减弱其发射荧光的强度 ,但对其λmax没有影响 .通过测定不同Ca2 +浓度下荧光强度的变化 ,可计算出在无Mg2 +存在时 ,该蛋白质的钙结合常数为 3.2× 10 - 8mol/L ,而在Mg2 +饱和条件下 ,钙结合常数为 7.4× 10 - 6 mol/L .乙醇亦不影响该蛋白质荧光的λmax,而对其荧光强度的影响则依赖于是否与钙结合 .未与Ca2 +结合时 ,乙醇浓度的增加导致荧光强度增强 ,与Ca2 +结合后 ,在低浓度乙醇溶液中 ,蛋白荧光强度减小 ,至乙醇浓度大于 16 .6 %后 ,其荧光强度逐渐增强 ,在 35 %乙醇溶液中 5 4kDCaBP对Ca2 +不敏感     

麦冬凝集素的氨基酸修饰和荧光光谱研究

通过对麦冬凝集素（OJL）进行特殊氨基酸的化学修饰，揭示了OJL所含的6个Trp残基只有1个位于蛋白表面，Trp、Tyr和Ser/Thr不是OJL凝集活性所必需的氨基酸，而Arg是维持其活性的必需基团.OJL在天然状态下荧光发射峰在328 nm处，Trp周围的极性较弱，处于疏水的微环境或Trp的吲哚环有特殊的构象.丙烯酰胺可以淬灭93.46%的色氨酸荧光，而CsCl和KI淬灭的程度较小，分别为41.25%和55.56%，证明Trp主要位于疏水环境.     

乙醇对鲍鱼碱性磷酸酶活力与构象的影响

以乙醇为效应物研究对鲍鱼碱性磷酸酶(ALP)活力影响的结果表明．酶的剩余活力随着乙醇浓度增大而迅速下降，乙醇浓度40％可使酶活力完全丧失．说明乙醇对鲍鱼ALP有明显的失活作用，JG50为13％．含较低浓度乙醇(30％)的失活过程是可逆的反应．测定乙醇对酶的失活作用机理．结果表明乙醇对鲍鱼ALP的失活作用是非竞争性机制，说明底物存在不影响乙醇对酶的失活作用．应用荧光光谱、紫外吸收光谱研究鲍鱼ALP经乙醇微扰后的分子构象变化，发现乙醇对酶分子构象有显的影响，酶的内源荧光强度随乙醇浓度增大而增强．荧光发射峰逐渐发生红移．紫外吸收光谱在276nm吸收峰随乙醇浓度增大而增强．这些结果表明．酶蛋白分子中的生色基团残基的微环境发生变化．     

曲酸与胰蛋白酶相互作用的光谱学研究

研究了曲酸对胰蛋白酶活性的影响,以及曲酸处理后胰蛋白酶紫外光谱、荧光淬灭光谱、同步荧光光谱和三维荧光光谱的变化．结果显示曲酸对胰蛋白酶具有激活作用;二者相互作用后,曲酸可以使胰蛋白酶的紫外吸收强度增强,但却使得蛋白内源荧光强度下降;Stern-Volmer作图显示曲酸对胰蛋白酶的荧光淬灭为静态淬灭作用;用同步荧光光谱和三维荧光光谱探讨了曲酸对胰蛋白酶构象的影响．     

碱脱胶法优化提取蚕丝素蛋白及其构象光谱

采取碱脱胶法对广西产桑蚕茧脱胶条件进行优化,利用苦味酸胭脂红法和扫描电镜对蚕丝脱胶程度进行检测,结果表明,蚕丝在脱胶浴比为1:200,脱胶30 min的条件下,其表面丝胶可脱除效果最好。将蚕丝脱胶所得丝素蛋白分别溶解在CaCl2-EtOH-H2O、LiBr-H_2O和LiBr-EtOH-H_2O3种溶剂中获再生蚕丝素蛋白溶液。在相同条件下,采用红外光谱、拉曼光谱对比研究蚕丝索蛋白的构象,结果表明碱脱胶法得到的天然丝素蛋白以β-折叠构象为主,再生丝素蛋白溶液构象以无规卷曲为主。对比3种溶剂对蛋白构象的影响,结果表明:溶剂中Li~(+)比Ca~(2+)对丝素蛋白的二级结构扰动更显著,倾向破坏蚕丝素蛋白的β-折叠构象,且溶剂中加入适量乙醇会进一步促进金属离子对丝素蛋白构象的影响。     

荧光光谱法研究绞股蓝皂苷与人血清白蛋白的相互作用

在pH=7.40的Tris-HCl缓冲溶液中,用荧光光谱法研究了绞股蓝皂苷与人血清白蛋白(HSA)相互作用的情况.结果表明,绞股蓝皂苷对人血清白蛋白的内源荧光有明显的猝灭作用,猝灭过程为动态猝灭.并计算得到绞股蓝皂苷与HSA的结合位点数和结合常数.通过热力学数据分析,推断出绞股蓝皂苷与人血清白蛋白之间主要靠疏水作用结合.同时采用同步荧光技术考察了绞股蓝皂苷对HSA构象的影响.     

弱光和Tris处理条件下PSⅡ放氧核心复合物的损伤

弱光条件下(120μmol·m-2·s-1)用Tris(0.8mol/L,pH6.5～10.0)处理具有放氧活性的PSⅡ核心复合物,可引起33kD锰稳定蛋白的释放和锰复合物的破坏,并导致核心复合物的结构发生明显的改变.温和电泳、SDS-PAGE和双向电泳分析表明,主要是PSⅡ核心复合物的二聚体和单体减少,并且复合物部分解体;除反应中心D1和D2蛋白外,核心天线CP43和CP7的量也减少,33kD锰稳定蛋白要发生降解.避光时,PSⅡ核心复合物不受影响.     

Low pH-induced conformational changes in 33 kD protein of photosystem Ⅱ

33 kD protein, located on the lumen side ofthylakoid membranes, is one of three extrinsic proteins ofphotosystem Ⅱ (PS Ⅱ ). Previous study showed that NBSmodification of W241, the only tryptophan in 33 kD protein,is helpful for understanding the function of W241 in main-taining functional conformation of 33 kD protein. In thispaper, studies of both circular dichroism and fluorescencespectra showed that upon decreasing pH from 6.2 to 2.5, theconformation of soluble 33 kD protein changed significantly,with an increase or a decrease in percentage of random coilor (z-helix and turns. The changes in secondary structures ofthis protein are pH reversible. After NBS modification at pH2.5, the conformational change of 33 kD protein was keptfixed. The CD ellipticity at 200 nm for NBS-modified 33 kDprotein is much lower than that for control, indicating that the unfolding degree of 33 kD protein was enhanced after the NBS modification. Moreover, the conformational flexibility islost in NBS-modified 33 kD protein, and the conformationalchange becomes pH irreversible, indicating that NBS modi-fication blocked the reversibility of conformational change of33 kD protein. The specific binding capability of NBS-modi-fled 33 kD protein is much lower than that of low pH-treatedcontrol. Furthermore, the rebinding of modified protein on PS Ⅱ membranes cannot restore the activity of oxygen evo-lution. We suggest that it is low pH but not NBS modificationof W241 that leads to the conformational change of 33 kDprotein from one functional to another non-functional state.The significant capability of proton transport of 33 kD pro-tein is discussed.     

Thermal stability of oxygen evolution in photosystem Ⅱ core complex in the presence of digalactosyl diacylglyceroli

The influence of digalactosyldiacylglycerol (DGDG), one of the photosynthetic membrane lipids, on heat inactivation of the process of oxygen evolution has been studied in vitro in photosystem Ⅱ (PSⅡ) core complex. It was found that the temperature of semi-inactivation of oxygen evolution in the complex increased from 40.0 to about 43.0℃ in the presence of DGDG with 5-min heat treatment in the dark. Furthermore, when PSⅡ core complex was incubated for 5 min at 45.0℃, the oxygen evolution in the complex was completely lost, whilst the DGDG-complexed PSⅡ core complex still retained a 16% of activity (100% for 25.0℃). In addition, a 1-h incubation at 38.0℃ inactivated absolutely the oxygen evolution for the PSⅡ core complex. By contrast, there remained about 20% of activity (zero time for 100%) for the complex in the presence of DGDG under the same condition. These results indicate a new role of DGDG in the protection of PSⅡ core complex against the deleterious effects of temperature. It was most likely that DGDG-mediated stability toward thermal denaturation of oxygen evolution in PSⅡ core complex is due to the protective effect of DGDG on the release of the 33 kD protein from PSⅡ core complex.     

Light and Heat Induced Denaturation of Photosystem Ⅱ Core Antenna Complex CP47

Light and heat induced denaturation of CP47, the core antenna complex of photosystem Ⅱ purified from spinach, were investigated using absorption and circular dichroism spectra.Light caused the destruction of chlorophyll a and excitonic interaction of chlorophyll a in CP47, while the protein secondary structure was not apparently changed.Heat induced the destruction of protein secondary structure and excitonic interaction of chlorophyll a, but the chlorophyll a molecule was not damaged.The results suggest that both the chlorophyll a molecular structure and the protein native conformation are necessary for excitonic interaction of chlorophyll a and the energy transfer function of the chlorophyll a binding protein.     

Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II

Oxygenic photosynthesis in plants, algae and cyanobacteria is initiated at photosystem II, a homodimeric multisubunit protein-cofactor complex embedded in the thylakoid membrane. Photosystem II captures sunlight and powers the unique photo-induced oxidation of water to atmospheric oxygen. Crystallographic investigations of cyanobacterial photosystem II have provided several medium-resolution structures (3.8 to 3.2 A) that explain the general arrangement of the protein matrix and cofactors, but do not give a full picture of the complex. Here we describe the most complete cyanobacterial photosystem II structure obtained so far, showing locations of and interactions between 20 protein subunits and 77 cofactors per monomer. Assignment of 11 beta-carotenes yields insights into electron and energy transfer and photo-protection mechanisms in the reaction centre and antenna subunits. The high number of 14 integrally bound lipids reflects the structural and functional importance of these molecules for flexibility within and assembly of photosystem II. A lipophilic pathway is proposed for the diffusion of secondary plastoquinone that transfers redox equivalents from photosystem II to the photosynthetic chain. The structure provides information about the Mn4Ca cluster, where oxidation of water takes place. Our study uncovers near-atomic details necessary to understand the processes that convert light to chemical energy.     

Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein

The Rho-family GTPase, Cdc42, can regulate the actin cytoskeleton through activation of Wiskott-Aldrich syndrome protein (WASP) family members. Activation relieves an autoinhibitory contact between the GTPase-binding domain and the carboxy-terminal region of WASP proteins. Here we report the autoinhibited structure of the GTPase-binding domain of WASP, which can be induced by the C-terminal region or by organic co-solvents. In the autoinhibited complex, intramolecular interactions with the GTPase-binding domain occlude residues of the C terminus that regulate the Arp2/3 actin-nucleating complex. Binding of Cdc42 to the GTPase-binding domain causes a dramatic conformational change, resulting in disruption of the hydrophobic core and release of the C terminus, enabling its interaction with the actin regulatory machinery. These data show that 'intrinsically unstructured' peptides such as the GTPase-binding domain of WASP can be induced into distinct structural and functional states depending on context.     

Depletion of phosphatidyl-glycerol head-group induces changes in oxygen evolution and protein secondary struc-tures of photosystem Ⅱ

The techniques of oxygen electrode polarogra-phy and Fourier transform infrared (FT-IR) spectroscopy were employed to explore the roles of polar head-group of phosphatidylglycerol (PG) molecules in the functional and structural aspects of photosystem Ⅱ (PS Ⅱ) through enzymatic approach. It was shown that the depletion of PG by treatment of phospholipase C (PLC) on PS Ⅱ particles caused the inhibition of oxygen evolving activity in PS Ⅱ. This effect also gave rise to changes in the protein secondary structures of PS Ⅱ, that is, an increase in a-helical conformation which is compensated by the loss of p-strand structures. It revealed that the head-group of PG molecules plays an important structural role in the maintenance of normal structure of PS Ⅱ proteins, which is required to maintain the appropriate physiological activity of the PS Ⅱ complex such as the oxygen evolving activity. It is suggested that there most probably exist hydrogen-bonding interactions between PG molecules and PS Ⅱ proteins.     

Trichloroacetate affects the EPR Signal IIslow and Signal I in the thylakoid ofChlamydomonas reinhardtii

One electron paramagnetic resonance (EPR) signal, named Signal II_slow, originates from the oxidized Tyrosine 160 (Y_D) of D2 polypeptide of photosystem II reaction center. After adding high concentration trichloroacetate (TCA) to theChlamydomonas reinhardtii thylakoid suspension, this signal was abolished in a minute. Treatment of TCA also removes a few of polypeptides, including three extrinsic polypeptides of oxygen-evolving complex, from the thylakoid membrane. Based upon the analysis of the microenvironment around Y_D with a three-dimensional model, it is indicated that relatively high hydrophobicity of this microenvironment may be the essential prerequisite for TCA to affect Y_D. It has been observed that TCA treatment also retards the decay of the Signal 1, produced by the oxidized reaction center chlorophyll dimer (P700⁺) of photosystem I.     

Crystal structure of plant photosystem I

Oxygenic photosynthesis is the principal producer of both oxygen and organic matter on Earth. The conversion of sunlight into chemical energy is driven by two multisubunit membrane protein complexes named photosystem I and II. We determined the crystal structure of the complete photosystem I (PSI) from a higher plant (Pisum sativum var. alaska) to 4.4 A resolution. Its intricate structure shows 12 core subunits, 4 different light-harvesting membrane proteins (LHCI) assembled in a half-moon shape on one side of the core, 45 transmembrane helices, 167 chlorophylls, 3 Fe-S clusters and 2 phylloquinones. About 20 chlorophylls are positioned in strategic locations in the cleft between LHCI and the core. This structure provides a framework for exploration not only of energy and electron transfer but also of the evolutionary forces that shaped the photosynthetic apparatus of terrestrial plants after the divergence of chloroplasts from marine cyanobacteria one billion years ago.     

家蚕卵蛋白的 SDS-PAGE 分析(动物科学)

本研究探讨了蛋白质提取方法、十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)条件对家蚕卵蛋白分离效果的影响。分别用裂解液提取法、TCA沉淀法、冷丙酮沉淀法和TCA-丙酮沉淀法制备蛋白质样品,然后进行6%～12%、6%～15%两种梯度胶的SDS-PAGE,获得了不同提取方法和电泳条件下的电泳图谱用以比较家蚕卵蛋白分离效果。结果表明用TCA-丙酮沉淀法除盐,再用含9 mol.L-1尿素、4%CHAPS、1%DTT的裂解液重悬,最适合家蚕卵蛋白提取;6%～12%梯度胶对家蚕卵蛋白的分离较为适宜。利用这一优化方法对家蚕感染微孢子虫的蚕卵和正常蚕卵进行了比较研究,在分子量66 kD和85 kD处找到了与感染微孢子虫相关的蛋白,为进一步进行正常蚕卵和感染微孢子虫蚕卵的蛋白质组学研究奠定了基础。