Membrane-mediated assembly of filamentous bacteriophage Pf1 coat protein

Filamentous bacteriophage Pf1 assembles by a membrane-mediated process during which the viral DNA is secreted through the membrane while being encapsulated by the major coat protein. Neutron diffraction studies showed that in the virus most of the coat protein consists of two alpha-helical segments arranged end-to-end with an intervening mobile surface loop. Nuclear magnetic resonance studies of the coat protein in the membrane-bound form have shown that the secondary structure is essentially identical to that in the intact virus. A comparison indicates that during membrane-mediated viral assembly, while the secondary structure of the coat protein is largely conserved, its tertiary structure changes substantially.     

New tools provide new insights in NMR studies of protein dynamics

There is growing evidence that structural flexibility plays a central role in the function of protein molecules. Many of the experimental data come from nuclear magnetic resonance (NMR) spectroscopy, a technique that allows internal motions to be probed with exquisite time and spatial resolution. Recent methodological advancements in NMR have extended our ability to characterize protein dynamics and promise to shed new light on the mechanisms by which these molecules function. Here, we present a brief overview of some of the new methods, together with applications that illustrate the level of detail at which protein motions can now be observed.     

Three-dimensional structure of the adenovirus major coat protein hexon

The three-dimensional crystal structure of the adenovirus major coat protein is presented. Adenovirus type 2 hexon, at 967 residues, is now the longest polypeptide whose structure has been determined crystallographically. Taken with our model for hexon packing, which positions the 240 trimeric hexons in the capsid, the structure defines 60% of the protein within the 150 X 10(6) dalton virion. The assembly provides the first details of a DNA-containing animal virus that is 20 times larger than the spherical RNA viruses previously described. Unexpectedly, the hexon subunit contains two similar beta-barrels whose topology is identical to those of the spherical RNA viruses, but whose architectural role in adenovirus is very different. The hexon structure reveals several distinctive features related to its function as a stable protective coat, and shows that the type-specific immunological determinants are restricted to the virion surface.     

Genetic coding: oligonucleotide coding for first six amino acid residues of the coat protein of R17 bacteriophage

Ribonucleic acids from the bacteriophage R17 and from R17 amber mutant AmB2 have been digested with ribonuclease T1. Of the products isolated, only one was different. It codes for the first six amino acid residues of the viral coat protein. The probable base sequence of the wild-type oligonucleotide is CpUpUpCpUpApApCpUpUpUpApCpUpCpApGp.     

奶牛红毛基因分型及蛋白结构预测研究

【目的】通过对奶牛MC1R基因核苷酸序列的分析和蛋白结构的预测,探讨奶牛红白花毛色形成的分子机制。【方法】采用PCR-RFLP技术对奶牛MC1R基因进行分型,利用生物信息学方法对其蛋白结构特征进行模拟分析。【结果】共检测出与毛色表型有关的3种基因型,其中EE基因型在黑白花奶牛中占优势,ee基因型在红白花奶牛中占优势。蛋白结构预测结果显示,ee基因型个体的缺失突变使蛋白质翻译提前终止,进而引发二级结构改变,导致红白花奶牛个体的出现。【讨论】E等位基因主要与黑色毛色形成有关,e等位基因主要与红色毛色形成有关。     

NMR structure of Mistic, a membrane-integrating protein for membrane protein expression

Although structure determination of soluble proteins has become routine, our understanding of membrane proteins has been limited by experimental bottlenecks in obtaining both sufficient yields of protein and ordered crystals. Mistic is an unusual Bacillus subtilis integral membrane protein that folds autonomously into the membrane, bypassing the cellular translocon machinery. Using paramagnetic probes, we determined by nuclear magnetic resonance (NMR) spectroscopy that the protein forms a helical bundle with a surprisingly polar lipid-facing surface. Additional experiments suggest that Mistic can be used for high-level production of other membrane proteins in their native conformations, including many eukaryotic proteins that have previously been intractable to bacterial expression.     

Intercalation complex of proflavine with DNA: structure and dynamics by solid-state NMR

The structure of the complex formed between the intercalating agent proflavine and fibrous native DNA was studied by one- and two-dimensional high-resolution solid-state nuclear magnetic resonance (NMR). Carbon-13-labeled proflavine was used to show that the drug is stacked with the aromatic ring plane perpendicular to the fiber axis and that it is essentially immobile. Natural abundance carbon-13 NMR of the DNA itself shows that proflavine binding does not change the puckering of the deoxyribose ring. However, phosphorus-31 NMR spectra show profound changes in the orientation of the phosphodiester grouping on proflavine binding, with some of the phosphodiesters tilting almost parallel to the helix axis, and a second set almost perpendicular. The first group to the phosphodiesters probably spans the intercalation sites, whereas the tilting of the second set likely compensates for the unwinding of the DNA by the intercalator.     

HIV-1 coat protein neurotoxicity prevented by calcium channel antagonists

Coat protein gp120 from the human immunodeficiency virus type-1 (HIV-1) increased intracellular free calcium and injured rodent retinal ganglion cells and hippocampal neurons in culture. Highly purified recombinant gp120 envelope protein produced these effects in a dose-dependent fashion at picomolar concentrations. Immunoprecipitation with antibody to gp120, but not with control immunoglobulin-containing serum, depleted solutions of the viral envelope protein and also prevented both the rise in intracellular calcium and neuronal toxicity. The gp120-induced increase in intracellular calcium was abrogated by transiently lowering extracellular calcium or by adding the dihydropyridine calcium channel antagonist nimodipine (100 nM). Calcium channel antagonists also prevented gp120-induced neuronal injury. In addition, intracellular stores appeared to contribute substantially to the increase in calcium elicited by gp120. Since increases in intracellular calcium have been associated with neurotoxicity, it is possible that an injurious effect of gp120 on neurons might be related to this mechanism and that treatment with calcium channel antagonists may prove useful in mitigating HIV-1-related neuronal injury.     

Determination of membrane protein structure by rotational resonance NMR: bacteriorhodopsin

Rotationally resonant magnetization exchange, a new nuclear magnetic resonance (NMR) technique for measuring internuclear distances between like spins in solids, was used to determine the distance between the C-8 and C-18 carbons of retinal in two model compounds and in the membrane protein bacteriorhodopsin. Magnetization transfer between inequivalent spins with an isotropic shift separation, delta, is driven by magic angle spinning at a speed omega r that matches the rotational resonance condition delta = n omega r, where n is a small integer. The distances measured in this way for both the 6-s-cis- and 6-s-trans-retinoic acid model compounds agreed well with crystallographically known distances. In bacteriorhodopsin the exchange trajectory between C-8 and C-18 was in good agreement with the internuclear distance for a 6-s-trans configuration [4.2 angstroms (A)] and inconsistent with that for a 6-s-cis configuration (3.1 A). The results illustrate that rotational resonance can be used for structural studies in membrane proteins and in other situations where diffraction and solution NMR techniques yield limited information.     

P1 of strawberry vein banding virus,a multilocalized protein,functions as a movement protein and interacts with the coat protein

Although the complete nucleotide sequence of strawberry vein banding virus (SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of each protein is unclear. This study provided evidence that the P1 protein of SVBV (SVBV-P1) possesses the following features. Bioinformatic and subcellular localization analyses showed that SVBV-P1 is localized in the cytoplasm and cell walls of epidermal cells in Nicotiana benthamiana, and it forms inclusion bodies associated with microtubules and the endoplasmic reticulum. Dilution experiments demonstrated that SVBV-P1 could move from the original agro-infiltrated cells to adjacent cells in N. benthamiana leaves. Further trans-complementation experiments demonstrated that SVBV-P1 could facilitate the intercellular movement of a movement-deficient potato virus X mutant in N. benthamiana leaves. Finally, yeast two-hybrid and bimolecular fluorescence complementation assays revealed that SVBV-P1 could interact with the SVBV coat protein, which is a major component of Caulimovirus virions. Results of the electrophoretic mobility shift assay indicated that SVBV-P1 lacks DNA-binding capability. In summary, the results suggest that SVBV-P1 is probably a movement protein of SVBV, providing new insights into the function of movement proteins of the Caulimovirus genus.     

Ultrastructural studies of seed coat and cotyledon during rapeseed maturation

Brassica napus L.(B. napus) is an important oil crop worldwide and it rapidly accumulates oil at late stage of seed maturation. However, little is known about the cellular mechanism of oil accumulation and seed color changes during the late stage of rapeseed development. Here, we analyzed the ultrastructure of seed coat, aleurone and cotyledon in embryos of B. napus from 25 to 70 days after flowering(DAF). The pigments, which were deposited on the cell wall of palisade cells in seed coat, determined dark black color of rapeseed. The chloroplasts degenerated into non-photosynthetic plastids which caused the green cotyledon to turn into yellow. The chloroplasts in aleurone and cotyledon cells respectively degenerated into remnants without inner and outer envelope membranes and ecoplasts with intact inner and outer envelope membranes. From 40 to 70 DAF, there were degraded chloroplasts without thylakoid, oil bodies contacting with plastids or protein bodies, big starch deposits of chloroplasts degrading into small particles then disappearing, and small endoplasmic reticulum(ER) in aleurone and cotyledon cells. Additionally, there were decreases of chlorophyll content and dramatic increases of oil content in rapeseed. These results suggested that the rapid oil accumulation was independent on the NADPH synthesized by photosynthesis of chloroplasts and probably utilized other sources of reductant, such as the oxidative pentose phosphate pathway during the late stage of rapeseed development. The triacylglycerol assembly presumably utilizes the enzymes in the plastid, cytosol or oil body of cotyledon and aleurone cells.     

三倍体西瓜种子萌发障碍与种皮的关系

以三倍体西瓜‘黑牛’和二倍体西瓜‘黑宝’种子为材料,对种子形态指标、种皮的透性和种皮电镜超微结构进行了分析。试验结果:三倍体西瓜种子中种皮平均厚度约为二倍体西瓜种子的2倍,而且三倍体西瓜种子比二倍体多1个细胞排列非常致密的硬化组织;三倍体西瓜种子内种皮平均厚度约为二倍体的10倍,三倍体西瓜种子内种皮木质化,结构明显分为3层;在种子吸胀萌发过程中,三倍体和二倍体种子内种皮结构变化的差异非常明显,表明三倍体西瓜种子的中种皮和内种皮在一定程度上均阻碍种胚与外界的气体交换,影响种子的萌发,而二倍体西瓜种子的种皮对气体交换的影响不明显。     

水稻条纹病毒外壳蛋白基因和病害特异性蛋白基因的克隆和序列分析

水稻条纹病毒编码的外壳蛋白 (CP)和病害特异性蛋白 (SP)是两种与症状密切相关的蛋白 .本文对我国云南 YL分离物的 CP基因和 SP基因进行了克隆和测序 .结果表明 ,CP基因和 SP基因分别由 96 6和 534个核苷酸组成 .与已发表的一些分离物相比 ,YL分离物 CP基因与我国山东 C分离物和日本 T、M分离物的核苷酸序列一致性在 96 .0 %左右 ,氨基酸序列一致性为 97.0 % ,但从序列一致性和碱基变异方式来看 ,C分离物与 T、M分离物的亲缘关系稍近 .YL 分离物 SP基因和云南 CX分离物之间有 99.0 %的核苷酸序列一致性 ,但与 T、M分离物的核苷酸一致性只有 94 .0 %左右 .可见 ,不同分离物间 SP基因的变异与其地理分布有密切的关系 .     

芜菁花叶病毒杭州分离株外壳蛋白基因序列分析

从杭州郊区分离出一个ＴｕＭＶ强毒株系，利用分子克隆和Ｓａｎｇｅｒ双脱氢测序方法分析了其外壳蛋白基因的ＤＮＡ序列．该序列与已报道的其他４种ＴｕＭＶ分离物都具有较高的同源性，最高达９７．６％，最低达８９．５％，其氨基酸序列的同源性更高，最高达９７．９％，最低达９４．５％。本文分析了该序列中ＡＴ和ＧＣ含量，计算了４种核苷酸在有意义链中和在密码子不同位置上的出现频率，同时还分析了该病毒外壳蛋白氨基酸遗传密码的使用频率。     

水稻条纹病毒外壳蛋白和病害特异蛋白在寄主体内的积累

ＰＡＳ ＥＬＩＳＡ检测结果表明：（１）水稻条纹病毒外壳蛋白和病害特异蛋白在水稻寄主体内累积量的变化趋势是一致的,而且均与寄主症状的严重度密切相关．（２）不同水稻品种中,２种蛋白的累积量和累积速率有明显差异．明恢６３（高感）２种蛋白的累积量均比ＩＲ３６（高抗）的明显大;０６３８１（耐受性低）２种蛋白的累积速率均比岗优２２（耐受性高）的明显快,０６３８１病叶中２种蛋白累积量在其显症３０ｄ左右达到高峰,而岗优２２的则在４０ｄ左右     

马铃薯卷叶病毒CP基因的突变及其原核表达

通过人工合成DNA的方法,对马铃薯卷叶病毒外壳蛋白(CP)基因第52～177核苷酸(126bp)这段序列进行了突变,将其中12个AGA、AGG、CGA等精氨酸稀有密码子变成了原核高效表达的同义密码子CGT与CGC,将另外两个AGA精氨酸密码子变成了错义密码。构建了突变基因的原核表达载体pBAD-LRCP2,Bsp1407Ⅰ与MssⅠ酶切及DNA测序结果表明,基因突变符合要求,表达载体的构建正确。在37℃,大肠杆菌工程株TOP10(pBAD-LRCP2)用0.2%L-阿拉伯糖诱导培养4h,SDS-PAGE显示蛋白图谱上有一条36ku的诱导表达的融合蛋白条带,结果表明突变基因在PBAD启动子驱动下在大肠杆菌中实现了表达。     

马铃薯A病毒云南分离物外壳蛋白基因的克隆与序列分析

根据马铃薯病毒A(Potato virusA,PVA)外壳蛋白(CP)基因序列设计合成的一对引物,以带病毒植株总RNA为模板,RT-PCR扩增得到长约800 bp的目的片段。将目的片段克隆至pGEM-T Easy载体并进行了序列测定,测得全长为807 bp的PVA CP基因。测序结果与PVA其他分离物CP基因序列比较,其氨基酸同源性最高可达98.5%。根据GenBank中PVA CP氨基酸序列建立了病毒的系统进化树并对PVA不同分离物CP氨基酸序列差异性进行了分析。     

大麦黄矮病毒外壳蛋白基因和移动蛋白基因酵母表达载体的构建

根据已知的大麦黄矮病毒GPV株系的外壳蛋白(Coat protein,CP)和移动蛋白(Movement protein,MP)基因序列合成了CP和MP基因的上下游引物,通过PCR扩增获得目的片段,经过Sal I和Pst I双酶切、连接、转化、重组质粒的酶切鉴定及基因测序,构建了酵母表达载体pGBKT7-GPV-CP和pGBKT7-GPV-MP,用于在酵母双杂交分析中表达诱饵融合蛋白,为进一步筛选小麦cDNA文库内与大麦黄矮病毒互作的寄主因子和克隆寄主因子,以及其种类和功能打下坚实的基础.