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Thioredoxin reductase from the malaria mosquito Anopheles gambiae. 冈比亚疟蚊的硫氧还蛋白还原酶。
Pub Date : 2003-11-01 DOI: 10.1046/j.1432-1033.2003.03812.x
Holger Bauer, Stephan Gromer, Andrea Urbani, Martina Schnölzer, R Heiner Schirmer, Hans-Michael Müller

The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A. gambiae and functionally substituted by the thioredoxin system. The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide-->NADP+ + thioredoxin dithiol. The A. gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant. The predominant isoform, A. gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells. In redox titrations, the substrate A. gambiae thioredoxin-1 (Km=8.5 microm, kcat=15.4 s(-1) at pH 7.4 and 25 degrees C) was unable to oxidize NADPH-reduced A. gambiae thioredoxin reductase-1 to the fully oxidized state. This indicates that, in contrast to other disulfide reductases, A. gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state. The thioredoxin reductases of the malaria system were compared. A. gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P. falciparum, respectively. A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation. The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A. gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P. falciparum enzyme. These differences offer an interesting approach to the design of species-specific inhibitors. Notably, A. gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redox-active Cys-Cys sequence.

冈比亚按蚊是恶性疟原虫的重要传播媒介。全基因组分析显示,与黑腹果蝇一样,冈比亚果蝇中没有谷胱甘肽还原酶,功能上被硫氧还蛋白系统所取代。该系统的关键酶是硫氧还蛋白还原酶-1,这是一种含有fad的二聚体蛋白,每个亚基55.3 kDa,催化NADPH + H+ +硫氧还蛋白二硫->NADP+ +硫氧还蛋白二硫醇的反应。冈比亚疟蚊trxr基因位于X染色体上,为单拷贝;它代表三个剪接变异体编码两个细胞质变异体和一个线粒体变异体。在大肠杆菌中重组表达了优势亚型冈比亚比亚硫氧还蛋白还原酶-1,并与野生型酶进行了功能比较,最终产量为1.4 U.ml(-1)。在氧化还原滴定中,底物A. gambiae thioredoxin-1 (Km=8.5微米,kcat=15.4 s(-1), pH为7.4,25℃)不能将nadph还原的A. gambiae thioredoxin reducase -1氧化至完全氧化状态。这表明,与其他二硫还原酶不同,冈比亚硫氧还蛋白还原酶-1在催化过程中在四电子还原态和两电子还原态之间振荡。比较了疟疾系统的硫氧还蛋白还原酶。冈比亚拟虫硫氧还蛋白还原酶-1与人类和恶性疟原虫同源物序列同源性分别为52%和45%。这三种酶的主要区别是c端氧化还原中心的结构,反映在催化中间体对自氧化的不同抗性上。该中心的相关序列为冈比亚亚种硫氧还蛋白还原酶的Thr-Cys-Cys-SerOH,人硫氧还蛋白还原酶的gly - cys -硒半胱氨酸- glyoh,恶性疟原虫硫氧还蛋白还原酶的cys - x - x - x - cys - glyoh。这些差异为设计物种特异性抑制剂提供了一种有趣的方法。值得注意的是,冈比亚芽孢杆菌硫氧还蛋白还原酶-1不是一种硒酶,而是含有一个非常不寻常的氧化还原活性Cys-Cys序列。
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引用次数: 53
Beta-amyloid protein oligomers induced by metal ions and acid pH are distinct from those generated by slow spontaneous ageing at neutral pH. 金属离子和酸性pH诱导的β -淀粉样蛋白低聚物不同于中性pH下缓慢自发老化产生的低聚物。
Pub Date : 2003-11-01 DOI: 10.1046/j.1432-1033.2003.03815.x
Genevieve M J A Klug, Dusan Losic, Supundi S Subasinghe, Marie-Isabel Aguilar, Lisandra L Martin, David H Small

Amyloid protein (Abeta1-40) aggregation and conformation was examined using native and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the results compared with those obtained by atomic force microscopy, and with Congo red binding, sedimentation and turbidity assays. The amount of Abeta aggregation measured was different, depending upon the method used. Incubation for 15 min at pH 5.0 or in the presence of Fe2+, Cu2+ or Zn2+ did not alter the level of Abeta oligomers observed on SDS and native gels. However, the slow aggregation of Abeta to form high molecular mass species over 5 days was inhibited. In contrast, when Abeta aggregation was monitored using a Congo red binding assay or sedimentation assay, a rapid increase in Abeta aggregation was observed after incubation for 15 min at pH 5.0, or in the presence of Fe2+, Cu2+ or Zn2+. The low pH-, Zn2+- or Cu2+-induced Abeta aggregation measured in a turbidity assay was reversible. In contrast, a considerable proportion of the Abeta aggregation measured by native and SDS/PAGE was stable. Atomic force microscopy studies showed that Abeta aged at pH 5.0 or in the presence of Zn2+ produced larger looser rod-shaped aggregates than at pH 7.4. Abeta that had been aged at pH 7.4 was more cytotoxic than Abeta aged at pH 5.0. Taken together, the results suggest that Abeta oligomerizes via two mutually exclusive mechanisms to form two different types of aggregates, which differ in their cytotoxic properties.

用天然和十二烷基硫酸钠/聚丙烯酰胺凝胶电泳检测淀粉样蛋白(Abeta1-40)的聚集和构象,并将结果与原子力显微镜、刚果红结合、沉淀和浊度测定结果进行比较。根据使用的方法,测量的β聚集量是不同的。在pH 5.0或Fe2+, Cu2+或Zn2+存在下孵育15分钟,不改变SDS和天然凝胶上观察到的Abeta低聚物水平。然而,在5天内,Abeta缓慢聚集形成高分子质量的物种被抑制。相比之下,当使用刚刚红结合试验或沉降试验监测Abeta聚集时,在pH 5.0或Fe2+, Cu2+或Zn2+存在下孵育15分钟后,观察到Abeta聚集迅速增加。在浊度测定中测量的低pH, Zn2+或Cu2+诱导的β聚集是可逆的。相比之下,通过原生和SDS/PAGE测量的相当一部分Abeta聚集是稳定的。原子力显微镜研究表明,在pH 5.0或Zn2+存在下,Abeta比pH 7.4时产生更大更松散的棒状聚集体。pH为7.4时老化的Abeta比pH为5.0时老化的Abeta具有更大的细胞毒性。综上所述,结果表明,Abeta寡聚通过两种相互排斥的机制形成两种不同类型的聚集体,这两种聚集体的细胞毒性不同。
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引用次数: 101
Biochemical and molecular characterization of hazelnut (Corylus avellana) seed lipoxygenases. 榛子种子脂氧化酶的生化和分子特性研究。
Pub Date : 2003-11-01 DOI: 10.1046/j.1432-1033.2003.03831.x
Angelo Santino, Angelo De Paolis, Antonia Gallo, Angela Quarta, Rod Casey, Giovanni Mita

Plant lipoxygenases (LOXs) are a class of dioxygenases which display diverse functions in several physiological processes such as growth, development and response to biotic and abiotic stresses. Even though LOXs have been characterized from several plant species, the physiological role of seed LOXs is still unclear. With the aim to better clarify the occurrence of LOXs and their influence on hazelnut seed quality, we carried out the biochemical and molecular characterization of the main LOX isoforms expressed during seed development. A genomic clone containing a complete LOX gene was isolated and fully characterized. The 9887 bp sequence reported contains an open reading frame of 5334 bp encoding a putative polypeptide of 99 kDa. Semiquantitative RT-PCR carried out from RNAs extracted from seeds at different maturation stages showed that LOXs are mainly expressed at early developmental stages. These results were confirmed by LOX activity assays. Biochemical characterization of the reaction products of the hazelnut LOX indicated that it is a 9-LOX. Two cDNAs were isolated by RT-PCR carried out on total RNA from immature hazelnut seeds. Sequence analysis indicated that the two cDNAs are highly homologous (91.9% degree of identity) and one of these corresponded exactly to the genomic clone. The deduced amino acid sequences of the hazelnut LOXs showed that they are closely related to a previously reported almond LOX (79.5% identity) and, to a lesser extent, to some LOXs involved in plant responses to pathogens (cotton and tobacco LOXs, 75.5 and 74.6% identity, respectively). The physiological role of hazelnut LOXs and their role in influencing seed quality are also discussed.

植物脂氧化酶(LOXs)是一类双加氧酶,在植物的生长发育以及对生物和非生物胁迫的响应等生理过程中发挥着多种功能。尽管许多植物物种中都有lox的特征,但种子lox的生理作用尚不清楚。为了更好地阐明LOX的发生及其对榛子种子品质的影响,我们对种子发育过程中表达的主要LOX亚型进行了生化和分子表征。分离并鉴定了一个完整的LOX基因克隆。报道的9887 bp序列包含5334 bp的开放阅读框,编码99kda的推定多肽。对不同成熟期种子提取的rna进行半定量RT-PCR分析发现,LOXs主要在发育早期表达。这些结果被液态氧活性测定所证实。对反应产物的生化表征表明,该产物为9-LOX。利用RT-PCR对未成熟榛子种子的总RNA进行分离,得到2个cdna。序列分析表明,这两个cdna高度同源(同源度为91.9%),其中一个与基因组克隆完全对应。榛子LOX的氨基酸序列与先前报道的杏仁LOX(同源性为79.5%)密切相关,与一些参与植物对病原体反应的LOX(棉花和烟草LOX,同源性分别为75.5%和74.6%)有较低的亲缘关系。讨论了榛子LOXs的生理作用及其对种子品质的影响。
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引用次数: 29
The phosphorylation pattern of human alphas1-casein is markedly different from the ruminant species. 人α -酪蛋白的磷酸化模式与反刍动物明显不同。
Pub Date : 2003-09-01 DOI: 10.1046/j.1432-1033.2003.03755.x
Esben S Sørensen, Lise Møller, Maria Vinther, Torben E Petersen, Lone K Rasmussen

Caseins are highly phosphorylated milk proteins assembled in large colloidal structures termed micelles. In the milk of ruminants, alphas1-casein has been shown to be extensively phosphorylated. In this report we have determined the phosphorylation pattern of human alphas1-casein by a combination of matrix-assisted laser desorption mass spectrometry and amino acid sequence analysis. Three phosphorylation variants were identified. A nonphosphorylated form, a variant phosphorylated at Ser18 and a variant phosphorylated at Ser18 and Ser26. Both phosphorylation sites are located in the amino acid recognition sequence of the mammary gland casein kinase. Notably, no phosphorylations were observed in the conserved region covering residues Ser70-Glu78, which is extensively phosphorylated in the ruminant alphas1-caseins.

酪蛋白是高度磷酸化的牛奶蛋白,组装在称为胶束的大胶体结构中。在反刍动物的乳汁中,α - 1酪蛋白已被证明被广泛磷酸化。在本报告中,我们用基质辅助激光解吸质谱法和氨基酸序列分析相结合的方法确定了人α -酪蛋白的磷酸化模式。鉴定出三种磷酸化变异。一种非磷酸化的形式,一种变体在Ser18位点磷酸化,一种变体在Ser18和Ser26位点磷酸化。这两个磷酸化位点都位于乳腺酪蛋白激酶的氨基酸识别序列中。值得注意的是,在覆盖Ser70-Glu78残基的保守区域未观察到磷酸化,该区域在反刍动物α - 1酪蛋白中广泛磷酸化。
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引用次数: 25
Synthesis and characterization of Pi4, a scorpion toxin from Pandinus imperator that acts on K+ channels. 作用于K+通道的蝎毒Pi4的合成与表征。
Pub Date : 2003-09-01 DOI: 10.1046/j.1432-1033.2003.03743.x
Sarrah M'Barek, Amor Mosbah, Guillaume Sandoz, Ziad Fajloun, Timoteo Olamendi-Portugal, Hervé Rochat, François Sampieri, J Iñaki Guijarro, Pascal Mansuelle, Muriel Delepierre, Michel De Waard, Jean-Marc Sabatier

Pi4 is a 38-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the alpha KTX6 subfamily of short four-disulfide-bridged scorpion toxins acting on K+ channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme-based cleavage of synthetic Pi4 (sPi4) indicated half-cystine pairings between Cys6-Cys27, Cys12-32, Cys16-34 and Cys22-37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD50 of 0.2 microg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC50 of 8 pm) and Shaker B (IC50 of 3 nm) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125I-labeled apamin for binding to small-conductance Ca(2+)-activated K+ (SK) channels from rat brain synaptosomes (IC50 value of 0.5 microm). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked (BIGGER program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the interaction and the associated blockage of the Kv1.2 channel.

Pi4是一种由四个二硫桥交联的38个残留物毒素,从Chactidae scorpion Pandinus imperator的毒液中分离出来。Pi4与maurotoxin、Pi1、Pi7和HsTx1一起,属于作用于K+通道的四二硫短桥蝎子毒素α KTX6亚家族。由于其在毒液中的丰度很低,为了更好地表征其药理学和结构特性,我们化学合成了Pi4。对合成Pi4 (sPi4)的酶切表明,在Cys6-Cys27、Cys12-32、Cys16-34和Cys22-37之间存在半胱氨酸配对,表明这是一种传统的蝎子毒素网状结构(Pi1/HsTx1型)。体内小鼠脑室内注射sPi4具有致死性(LD50为0.2 μ g /只)。在体外,将sPi4添加到异种表达不同电压门控K+通道亚型的非洲爪蟾卵母细胞中,对大鼠Kv1.2通道(IC50为8 pm)和Shaker B通道(IC50为3 nm)的电流有明显的抑制作用,而对大鼠Kv1.1和Kv1.3通道没有影响。sPi4还被发现与125i标记的维生素a竞争,以结合大鼠脑突触体的小电导Ca(2+)激活的K+ (SK)通道(IC50值为0.5微米)。sPi4是Kv1.2通道的高亲和力阻滞剂。利用sPi4溶液结构和Kv1.2通道孔区分子模型,将毒素停靠在Kv通道上(BIGGER程序)。该模型表明,残基Arg10、Arg19、Lys26 (dyad)、Ile28、Lys30、Lys33和Tyr35 (dyad)在相互作用和相关的Kv1.2通道阻断中起关键作用。
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引用次数: 44
The CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto. Functional and structural insights through disulfide structure analysis. 人类CRIPTO/FRL-1/CRYPTIC (CFC)结构域。通过二硫化物结构分析来了解功能和结构。
Pub Date : 2003-09-01 DOI: 10.1046/j.1432-1033.2003.03749.x
Susan F Foley, Herman W T van Vlijmen, Raymond E Boynton, Heather B Adkins, Anne E Cheung, Juswinder Singh, Michele Sanicola, Carmen N Young, Dingyi Wen

The disulfide structure of the CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto protein was determined by a combination of enzymatic and chemical fragmentation, followed by chromatographic separation of the fragments, and characterization by mass spectrometry and N-terminal sequencing. These studies showed that Cys115 forms a disulfide bond with Cys133, Cys128 with Cys149, and Cys131 with Cys140. Protein database searching and molecular modeling revealed that the pattern of disulfide linkages in the CFC domain of Cripto is the same as that in PARS intercerebralis major Peptide C (PMP-C), a serine protease inhibitor, and that the EGF-CFC domains of Cripto are predicted to be structurally homologous to the EGF-VWFC domains of the C-terminal extracellular portions of Jagged 1 and Jagged 2. Biochemical studies of the interactions of ALK4 with the CFC domain of Cripto by fluorescence-activated cell sorter analysis indicate that the CFC domain binds to ALK4 independent of the EGF domain. A molecular model of the CFC domain of Cripto was constructed based on the nuclear magnetic resonance structure of PMP-C. This model reveals a hydrophobic patch in the domain opposite to the presumed ALK4 binding site. This hydrophobic patch may be functionally important for the formation of intra or intermolecular complexes.

人类CRIPTO蛋白的CRIPTO/FRL-1/CRYPTIC (CFC)结构域的二硫结构是通过酶促和化学裂解相结合的方法确定的,随后对片段进行色谱分离,并通过质谱和n端测序进行表征。这些研究表明,Cys115与Cys133、Cys128与Cys149、Cys131与Cys140形成二硫键。蛋白质数据库检索和分子模型显示,Cripto的CFC结构域的二硫键模式与丝氨酸蛋白酶抑制剂PARS interbralis major Peptide C (PMP-C)中的二硫键模式相同,并且预计Cripto的EGF-CFC结构域在结构上与Jagged 1和Jagged 2的C端胞外部分的EGF-VWFC结构域同源。通过荧光活化细胞分选分析对ALK4与Cripto的CFC结构域相互作用的生化研究表明,CFC结构域独立于EGF结构域与ALK4结合。基于PMP-C的核磁共振结构,构建了Cripto的CFC结构域分子模型。该模型揭示了在假定的ALK4结合位点的相反区域有一个疏水斑块。这种疏水斑块可能对分子内或分子间复合物的形成具有重要的功能。
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引用次数: 20
First evidence of catalytic mediation by phenolic compounds in the laccase-induced oxidation of lignin models. 酚类化合物在漆酶诱导的木质素氧化模型中催化调解的第一个证据。
Pub Date : 2003-09-01 DOI: 10.1046/j.1432-1033.2003.03752.x
Francesca d'Acunzo, Carlo Galli

The sulfonephthalein indicator, phenol red, exhibits an unusually slow rate of oxidation by laccase from Poliporus pinsitus, in spite of the fact that it is a phenol and therefore a natural substrate for this phenoloxidase enzyme. Nevertheless, after prolonged exposure to laccase (24 h) phenol red is oxidized by more than 90%. We found that phenol red, which can be oxidatively converted into a resonance-stabilized phenoxy radical, performs as a mediator in the laccase-catalyzed oxidation of a nonphenolic substrate (4-methoxybenzyl alcohol) and also of a hindered phenol (2,4,6-tri-tert-butylphenol). In particular, phenol red was found to be at least 10 times more efficient than 3-hydroxyanthranilate (a reported natural phenolic mediator of laccase) in the oxidation of 4-methoxybenzyl alcohol. Other phenols, which do not bear structural analogies to phenol red, underwent rapid degradation and did not perform as laccase mediators. On the other hand, several variously substituted sulfonephthaleins, of different pK2 values, mediated the laccase catalysis, the most efficient being dichlorophenol red, which has the lowest pK2 of the series. The mediating efficiency of phenol red and dichlorophenol red was found to be pH dependent, as was their oxidation Ep value (determined by cyclic voltammetry). We argue that the relative abundance of the phenoxy anion, which is easier to oxidize than the protonated phenol, may be one of the factors determining the efficiency of a phenolic mediator, together with its ability to form relatively stable oxidized intermediates that react with the desired substrate before being depleted in undesired routes.

磺胺苯酞指示剂苯酚红,尽管它是一种苯酚,因此是这种酚氧化酶的天然底物,但它被来自pinsitus的漆酶氧化的速度却异常缓慢。然而,长时间暴露于漆酶(24小时)后,酚红被氧化超过90%。我们发现,酚红可以氧化转化为共振稳定的苯氧基自由基,在漆酶催化的非酚类底物(4-甲氧基苄基醇)和受阻酚(2,4,6-三叔丁基酚)的氧化中起中介作用。特别是,在4-甲氧基苄基醇的氧化过程中,发现酚红的效率至少是3-羟基苯甲酸酯(一种报道的漆酶的天然酚类介质)的10倍。其他与酚红结构不相似的酚类物质会迅速降解,不能作为漆酶介质。另一方面,几种不同取代的不同pK2值的磺苯酞介导了漆酶的催化作用,其中效率最高的是二氯酚红,其pK2值最低。发现酚红和二氯酚红的中介效率与pH有关,其氧化Ep值(由循环伏安法测定)也与pH有关。我们认为,相对丰富的苯氧阴离子比质子化的苯酚更容易氧化,可能是决定酚醛介质效率的因素之一,以及它形成相对稳定的氧化中间体的能力,这些中间体在以不需要的途径耗尽之前与所需的底物反应。
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引用次数: 103
Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein. 马铃薯羧肽酶抑制剂前序列对成熟蛋白折叠的影响分析。
Pub Date : 2003-09-01 DOI: 10.1046/j.1432-1033.2003.03754.x
Sílvia Bronsoms, Josep Villanueva, Francesc Canals, Enrique Querol, Francesc X Aviles

Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form.

蛋白质折叠在体内可受多种因素调节。虽然伴侣蛋白作为折叠催化剂,显示出广泛的底物特异性,但一些前肽特异性地促进了它们所结合的成熟蛋白的折叠。马铃薯羧肽酶抑制剂(Potato carboxypeptidase inhibitor, PCI)是一种含有27个残基n端和7个残基c端前区的前体蛋白,是一种具有39个残基的羧肽酶抑制剂。在这项工作中,成熟的PCI在体外的二硫偶联折叠已经与用N端前序列(ProNtPCI)或N端和c端前序列(ProPCI)延伸的相同蛋白质的折叠进行了比较,也与反式中的N端前序列(ProNt + PCI)进行了比较。在所有这些分子的折叠动力学或效率方面没有观察到显着差异。此外,在大肠杆菌中使用野生型PCI和三种具有或不具有n端前序列的PCI突变形式进行了体内折叠研究,这些突变先前已报道影响成熟形式的PCI折叠。每个结构向介质分泌的“原生样”形式的程度不受n端前序列存在的影响。这些结果表明,在体外和体内大肠杆菌中,PCI的折叠不依赖于n端前序列。然而,通过光谱、氢交换和质谱有限蛋白水解的结构分析表明,这种n端前序列能够在前体形式内折叠。
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引用次数: 11
Prevalent conformations and subunit exchange in the biologically active apoptin protein multimer. 生物活性凋亡蛋白多聚体的普遍构象和亚基交换。
Pub Date : 2003-09-01 DOI: 10.1046/j.1432-1033.2003.03750.x
Sirik R Leliveld, Mathieu H M Noteborn, Jan Pieter Abrahams

Recombinant, bacterially expressed apoptin protein induces apoptosis in human tumour cell lines but not in normal cells, mimicking the behaviour of ectopically expressed apoptin. Recombinant apoptin is isolated exclusively as a highly stable multimeric complex of 30-40 monomers, with little, if any, alpha-helical and beta-sheet structure. Despite its apparent disorder, multimeric apoptin is biologically active. Here, we present evidence that most of the apoptin moieties within the complex may well share a similar conformation. Furthermore, the multimer has extensive and uniform hydrophobic patches and conformationally stable domains. Only a small fraction of apoptin subunits can exchange between multimers under physiologically relevant conditions. These results prompt a model in which the apoptin multimer has a highly stable core of nonexchangeable subunits to which exchangeable subunits are attached through hydrophobic interactions. In combination with previous findings, our results lead us to propose that the stable core of apoptin is the biologically relevant structure.

重组,细菌表达的凋亡蛋白诱导人肿瘤细胞系凋亡,而不是正常细胞,模仿异位表达的凋亡蛋白的行为。重组细胞凋亡素是一种高度稳定的多聚复合物,由30-40个单体组成,几乎没有α -螺旋和β -片结构。尽管多聚体细胞凋亡蛋白明显紊乱,但它具有生物活性。在这里,我们提出的证据表明,复合体内的大多数凋亡蛋白片段可能具有相似的构象。此外,多聚体具有广泛而均匀的疏水斑块和构象稳定的结构域。在生理相关条件下,只有一小部分凋亡素亚基可以在多聚体之间交换。这些结果提示了一个模型,其中凋亡蛋白多聚体具有高度稳定的不可交换亚基核心,可交换亚基通过疏水相互作用附着在核心上。结合先前的研究结果,我们的结果使我们提出凋亡蛋白的稳定核心是生物学相关结构。
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引用次数: 13
Does different orientation of the methoxy groups of ubiquinone-10 in the reaction centre of Rhodobacter sphaeroides cause different binding at QA and QB? 球形红杆菌反应中心泛素-10甲氧基的不同取向是否导致QA和QB的结合不同?
Pub Date : 2003-09-01 DOI: 10.1046/j.1432-1033.2003.03746.x
André Remy, Rutger B Boers, Tatiana Egorova-Zachernyuk, Peter Gast, Johan Lugtenburg, Klaus Gerwert

The different roles of ubiquinone-10 (UQ10) at the primary and secondary quinone (QA and QB) binding sites of Rhodobacter sphaeroides R26 reaction centres are governed by the protein microenvironment. The 4C=O carbonyl group of QA is unusually strongly hydrogen-bonded, in contrast to QB. This asymmetric binding seems to determine their different functions. The asymmetric hydrogen-bonding at QA can be caused intrinsically by distortion of the methoxy groups or extrinsically by binding to specific amino-acid side groups. Different X-ray-based structural models show contradictory orientations of the methoxy groups and do not provide a clear picture. To elucidate if distortion of the methoxy groups induces this hydrogen-bonding, their (ring-)C-O vibrations were assigned by use of site-specifically labelled [5-13C]UQ10 and [6-13C]UQ10 reconstituted at either the QA or the QB binding site. Two infrared bands at 1288 cm(-1) and 1264 cm(-1) were assigned to the methoxy vibrations. They did not shift in frequency at either the QA or QB binding sites, as compared with unbound UQ10. As the frequencies of these vibrations and their coupling are sensitive to the conformations of the methoxy groups, different conformations of the C(5) and C(6) methoxy groups at the QA and QB binding sites can now be excluded. Both methoxy groups are oriented out of plane at QA and QB. Therefore, hydrogen-bonding to His M219 combined with electrostatic interactions with the Fe2+ ion seems to determine the strong asymmetric binding of QA.

泛素-10 (UQ10)在球形红杆菌R26反应中心一级和二级醌(QA和QB)结合位点的不同作用受蛋白质微环境的支配。与QB相比,QA的4C=O羰基具有异常强的氢键。这种不对称绑定似乎决定了它们的不同功能。QA上的不对称氢键可以由甲氧基的畸变引起,也可以由与特定氨基酸侧基的结合引起。不同的基于x射线的结构模型显示了甲氧基的相互矛盾的取向,并没有提供一个清晰的图像。为了阐明甲氧基的扭曲是否诱导了这种氢键,他们的(环)C-O振动通过使用位点特异性标记的[5-13C]UQ10和在QA或QB结合位点重组的[6-13C]UQ10来分配。在1288 cm(-1)和1264 cm(-1)的两个红外波段被分配给甲氧基振动。与未结合的UQ10相比,它们在QA或QB结合位点的频率都没有变化。由于这些振动的频率及其耦合对甲氧基的构象很敏感,现在可以排除QA和QB结合位点上C(5)和C(6)甲氧基的不同构象。两个甲氧基在QA和QB处都取向于平面外。因此,与His M219的氢键结合与Fe2+离子的静电相互作用似乎决定了QA的强不对称结合。
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引用次数: 12
期刊
European journal of biochemistry
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