高等植物Rubisco的组装及其中间产物的鉴定

将新鲜制备的不含Ｒｕｂｉｓｃｏ的水稻叶片低分子量蛋白组分在离体条件下于室温保温４８ｈ,ＮＤ－ＰＡＧＥ分析发现在ＡＴＰ ５ｍｍｏｌ／Ｌ和Ｍｇ＾２＋ ５ｍｍｏｌ／Ｌ的作用条件下,在分子量为５６０ｋＤ位置上有一蛋白带生成。高浓度的Ｋ拓一定程度上抑制它的形成,在保温介质中没有ＡＴＰ存在时,不能产生５６０ｋＤ分子量的条带,但有一更高分子量（约６００ｋＤ）蛋白条带的产生,这一条带能在ＡＴＰ和Ｍｇ＾２＋作用下发     

春天的脚步

三月是大地回暖、鸟语花香、春光明媚的季节,历经严冬的考验,春日的阳光照在身上倍觉温暖。在这里,我们满怀喜悦地告诉大家,在众多编委的大力支持下,截至二月底的统计显示,《学报》在2011年最初两个月中被引用的情况已经创造了历史最好记录。在过去十年中,《学报》     

真正的科学家真正的爱国者

卢永根院士是著名的作物遗传学家。他致力于水稻遗传育种研究,取得了显著的成绩。在青年时代,他以极大的热情投身到革命洪流中,既做地下党的工作,又刻苦地在岭大深造;在岭大毕业后至今50余年中,他在高校既教书,又从事科学研究,为人才培养和水稻遗传育种工作做出贡献。他总结的五点体会和崇高的思想境界:“我的青春年华已经献给党的科学事业,我准备把晚年继续献给这个事业。”令人深受启发,值得学习。经作者同意,现将刊登在中国科学院院士工作局《学部通讯》2003年第4期上的“院士自述:我的成长经历”这篇报道,转载于后。     

疫苗的历史

我们是怎样谈论人的？会不会像天文学家看到的那样只是一点尘埃，无依无靠地在一颗不重要的行星上蠕动？或像化学家所说的是巧妙地摆弄在一起的一堆化学品？或者像在哈姆雷特眼里看到的那样，人在理智上是高贵的，在才能上是无限的？或兼有以上的一切？     

摄影的精神

照相机．跟电脑和手机一样,都是当今世界最为普及的工具。事实上．现在有些照相机是与手机、电脑三者合一的。几乎每个人,至少住在城市里的每个人,都在拍照。至于说一个人的形象从来没有出现在照片上．这样的人几乎很难找到。在我们开汽车、逛商店或走在马路上时,我们的形象常常会在无意中被监控录像拍下来。     

公平贸易引发的是一连串的问号

<正>本期封面题目或许有些生僻,实则与世人皆不无关系。四川安龙村七户农民为成都部分消费者预定种植和配送生态蔬菜,贵州流芳村农民协会在全村推广种植和外销无证的有机稻,内蒙古阿拉善盟牧民合作社在努力把极干旱地区的稀品驼肉和驼绒推上市场,云南景迈山的古茶园在普洱茶价格暴涨暴跌中的奇特经历,贵州白兴村的手工刺绣在市场上的两难境遇,部分北京市民在郊区"小毛驴"农场当起菜农为自己种菜……类似本期所讲述的这些故事,想必在全国不止成百上千。而从这     

始祖鸟的故事

1860年在德国巴伐利亚索伦霍芬地区发现的一根羽毛化石,揭开了始祖鸟故事的序幕.这件羽毛压痕化石,在当时引起了轰动,它使得人们意识到在爬行动物横行的时代就有了鸟类的存在.     

淀粉合成受限的莱茵衣藻的甘油酯酰基的变化

【目的】基于突变藻株本身属性和意义出发,考察在两种常用培养方式下莱茵衣藻淀粉突变株(CC-4326)与野生型藻株(CC-137)在甘油酯中酰基随生长的变化差异,为进一步认识莱茵衣藻突变株提供参考信息。【方法】分别在柱状鼓泡式反应器和摇瓶中培养CC-4326和CC-137,比较两株藻在正常培养和氮胁迫培养状态下甘油酯中酰基相对含量和其在甘油三酯(TAG)含量的差异。【结果】正常培养状态下,CC-4326和CC-137中多不饱和酰基C16:4和C18:3相对含量占总酰基45%左右,CC-4326在两种培养方式下这两个酰基含量及变化无差别,而CC-137在摇瓶中培养二者相对含量增加幅度和含量均高于反应器。缺氮条件下两种藻株积累TAG,但程度不同,CC-4326在反应器中培养TAG含量达到CC-137的1.5倍,在摇瓶中培养含量与CC-137无显著差异,两株藻的甘油酯和TAG中C18:1含量显著增加,CC-4326在反应器中培养C18:1增加幅度大于摇瓶,比摇瓶培养更快速积累TAG。而CC-137在摇瓶中培养TAG含量与反应器接近,单不饱和酰基增加幅度却高于反应器,表明CC-137在摇瓶中培养比反应器更利于积累TAG。最终,CC-4326在光生物反应器中缺氮培养实现了TAG 12倍的增加。【结论】通过对淀粉合成抑制,与CC-137相比,缺氮光生物反应器培养条件下,CC-4326能够实现TAG的高效积累。     

没有神经的腿的再生

对于嵘螈肢体的再生,神经系统特别是切割后的残肢裹的神经纤维,一向是被认为有决定性意义的。晚近利用成体或接近变态的幼体的研究证明了:在再生芽基形成之后,切断神经,已经形成的芽基仍然可以继续发育成正常的肢体;但在再生芽基形成之     

带有特定染色体片段的mini-F质粒所发动的染色体复制对于recA基因的依赖性

构建了一些带有大肠杆菌的特定染色体片段的mini-F质粒,使它们通过同源重组整合在dnaA46细菌的染色体的预定位置上,然后测定整合抑制菌株(sin)在40℃中染色体复制对recA基因的依赖性。实验结果说明,Sin菌株对recA基因的依赖性决定在质粒的整合位置。整合在oriC近旁的Sin菌株不依赖于recA基因;整合在oriC和terC中间的只在丰富培养基上是依赖的;整合在rerC附近的在不丰富的培养基上也依赖于recA基因。     

Differential effects of co-chaperonin homologs on cpn60 oligomers

In this study, we have investigated the relationship between chaperonin/co-chaperonin binding, ATP hydrolysis, and protein refolding in heterologous chaperonin systems from bacteria, chloroplast, and mitochondria. We characterized two types of chloroplast cpn60 oligomers, ch-cpn60 composed of α and β subunits (α₇β₇ ch-cpn60) and one composed of all β subunits (β₁₄ ch-cpn60). In terms of ATPase activity, the rate of ATP hydrolysis increased with protein concentration up to 60 μM, reflecting a concentration at which the oligomers are stable. At high concentrations of cpn60, all cpn10 homologs inhibited ATPase activity of α₇β₇ ch-cpn60. In contrast, ATPase of β₁₄ ch-cpn60 was inhibited only by mitochondrial cpn10, supporting previous reports showing that β₁₄ is functional only with mitochondrial cpn10 and not with other cpn10 homologs. Surprisingly, direct binding assays showed that both ch-cpn60 oligomer types bind to bacterial, mitochondrial, and chloroplast cpn10 homologs with an equal apparent affinity. Moreover, mitochondrial cpn60 binds chloroplast cpn20 with which it is not able to refold denatured proteins. Protein refolding experiments showed that in such instances, the bound protein is released in a conformation that is not able to refold. The presence of glycerol, or subsequent addition of mitochondrial cpn10, allows us to recover enzymatic activity of the substrate protein. Thus, in our systems, the formation of co-chaperonin/chaperonin complexes does not necessarily lead to protein folding. By using heterologous oligomer systems, we are able to separate the functions of binding and refolding in order to better understand the chaperonin mechanism.     

Type I chaperonins: not all are created equal

Type I chaperonins play an essential role in the folding of newly translated and stress-denatured proteins in eubacteria, mitochondria and chloroplasts. Since their discovery, the bacterial chaperonins have provided an excellent model system for investigating the mechanism by which chaperonins mediate protein folding. Due to the high conservation of the primary sequence among Type I chaperonins, it is generally accepted that organellar chaperonins function similar to the bacterial ones. However, recent studies indicate that the chloroplast and mitochondrial chaperonins possess unique structural and functional properties that distinguish them from their bacterial homologs. This review focuses on the unique properties of organellar chaperonins.     

Location and flexibility of the unique C-terminal tail of Aquifex aeolicus co-chaperonin protein 10 as derived by cryo-electron microscopy and biophysical techniques

Co-chaperonin protein 10 (cpn10, GroES in Escherichia coli) is a ring-shaped heptameric protein that facilitates substrate folding when in complex with cpn60 (GroEL in E. coli). The cpn10 from the hyperthermophilic, ancient bacterium Aquifex aeolicus (Aacpn10) has a 25-residue C-terminal extension in each monomer not found in any other cpn10 protein. Earlier in vitro work has shown that this tail is not needed for heptamer assembly or protein function. Without the tail, however, the heptamers (Aacpn10del-25) readily aggregate into fibrillar stacked rings. To explain this phenomenon, we performed binding experiments with a peptide construct of the tail to establish its specificity for Aacpn10del-25 and used cryo-electron microscopy to determine the three-dimensional (3D) structure of the GroEL-Aacpn10-ADP complex at an 8-Å resolution. We found that the GroEL-Aacpn10 structure is similar to the GroEL-GroES structure at this resolution, suggesting that Aacpn10 has molecular interactions with cpn60 similar to other cpn10s. The cryo-electron microscopy density map does not directly reveal the density of the Aacpn10 25-residue tail. However, the 3D statistical variance coefficient map computed from multiple 3D reconstructions with randomly selected particle images suggests that the tail is located at the Aacpn10 monomer-monomer interface and extends toward the cis-ring apical domain of GroEL. The tail at this location does not block the formation of a functional co-chaperonin/chaperonin complex but limits self-aggregation into linear fibrils at high temperatures. In addition, the 3D variance coefficient map identifies several regions inside the GroEL-Aacpn10 complex that have flexible conformations. This observation is in full agreement with the structural properties of an effective chaperonin.     

Structural stability of oligomeric chaperonin 10: the role of two beta-strands at the N and C termini in structural stabilization

Chaperonin 10 (cpn10) is a well-conserved subgroup of the molecular chaperone family. GroES, the cpn10 from Escherichia coli, is composed of seven 10kDa subunits, which form a dome-like oligomeric ring structure. From our previous studies, it was found that GroES unfolded completely through a three-state unfolding mechanism involving a partly folded monomer and that this reaction was reversible. In order to study whether these unfolding-refolding characteristics were conserved in other cpn10 proteins, we have examined the structural stabilities of cpn10s from rat mitochondria (RatES) and from hyperthermophilic eubacteria Thermotoga maritima (TmaES), and compared the values to those of GroES. From size-exclusion chromatography experiments in the presence of various concentrations of Gdn-HCl at 25 degrees C, both cpn10s showed unfolding-refolding characteristics similar to those of GroES, i.e. two-stage unfolding reactions that include formation of a partially folded monomer. Although the partially folded monomer of TmaES was considerably more stable compared to GroES and RatES, it was found that the overall stabilities of all three cpn10s were achieved significantly by inter-subunit interactions. We studied this contribution of inter-subunit interactions to overall stability in the GroES heptamer by introducing a mutation that perturbed subunit association, specifically the interaction between the two anti-parallel beta-strands at the N and C termini of this protein. From analyses of the mutants' stabilities, it was revealed that the anti-parallel beta-strands at the subunit interface are crucial for subunit association and stabilization of the heptameric GroES protein.     

Kinetic folding and assembly mechanisms differ for two homologous heptamers

Here we investigate the time-resolved folding and assembly mechanism of the heptameric co-chaperonin protein 10 (cpn10) in vitro. The structure of cpn10 is conserved throughout nature: seven beta-barrel subunits are non-covalently assembled through beta-strand pairings in an overall doughnut-like shape. Kinetic folding/assembly experiments of chemically denatured cpn10 from Homo sapiens (hmcpn10) and Aquifex aeolicus (Aacpn10) were monitored by far-UV circular dichroism and fluorescence. We find the processes to be complex, involving several kinetic steps, and to differ between the mesophilic and hyper-thermophilic proteins. The hmcpn10 molecules partition into two parallel pathways, one involving polypeptide folding before protein-protein assembly and another in which inter-protein interactions take place prior to folding. In contrast, the Aacpn10 molecules follow a single sequential path that includes initial monomer misfolding, relaxation to productive intermediates and, subsequently, final folding and heptamer assembly. An A. aeolicus variant lacking the unique C-terminal extension of Aacpn10 displays the same kinetic mechanism as Aacpn10, signifying that the tail is not responsible for the rapid misfolding step. This study demonstrates that molecular details can overrule similarity of native-state topology in defining apparent protein-biophysical properties.     

Recombinant EPF/chaperonin 10 promotes the survival of O4-positive pro-oligodendrocytes prepared from neonatal rat brain

Chaperonin 10 (cpn 10) is a small heat-shock protein that is usually intracellular. Early pregnancy factor (EPF), a biologically active protein that was first described in the serum of pregnant mammals, is homologous to cpn 10. EPF/cpn 10 has been reported to have effects on immunomodulation and cell survival and to inhibit activation of toll-like receptors by lipopolysaccharide. We found that recombinant EPF/cpn 10 was able to suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, which is a disease causing inflammation and demyelination of the brain and spinal cord. This beneficial effect could be due to anti-inflammatory and/or cell survival properties of EPF/cpn 10. We aimed to assess the effects of cpn 10 on cells of the oligodendrocyte lineage because oligodendrocytes are the brain cells that produce myelin and that are depleted in multiple sclerosis. Two forms of recombinant EPF/cpn 10 were prepared in the pGEX expression system and in the baculovirus expression system. Purified O4⁺ pro-oligodendrocytes were prepared from the brains of day-old Wistar rats and isolated by cell sorting with flow cytometry. Single cells were dispensed into micro-well plates and tested for survival in the presence of a range of concentrations of the two forms of cpn 10. We also studied the effects of bFGF, PDGF, IGF-1 and insulin as controls. With cpn 10 present, there was enhanced survival of O4⁺ cells.     

Characterization of the cytoplasmic chaperonin containing TCP-1 from the Antarctic fish Notothenia coriiceps

The cytoplasmic chaperonin containing TCP-1 (CCT) plays a critically important role in the folding and biogenesis of many cytoskeletal proteins, including tubulin and actin. For marine ectotherms, the chronically cold Southern Ocean (−2 to +2°C) poses energetic challenges to protein folding, both at the level of substrate proteins and with respect to the chaperonin/chaperone folding system. Here we report the partial functional and structural characterization of CCT from an Antarctic notothenioid fish, Notothenia coriiceps. We find that the mechanism of folding by the Antarctic fish CCT differed from that of mammalian CCT: (1) the former complex was able to bind denatured β-tubulin but (2) when reconstituted with rabbit Cofactor A, failed to release the protein to yield the tubulin/cofactor intermediate. Moreover, the amino acid sequences of the N. coriiceps CCT β and θ chains contained residue substitutions in the equatorial, apical, and intermediate domains that would be expected to increase the flexibility of the subunits, thus facilitating function of the chaperonin in an energy poor environment. Our work contributes to the growing realization that protein function in cold-adapted organisms reflects a delicate balance between the necessity of structural flexibility for catalytic activity and the concomitant hazard of cold-induced denaturation.     

Mechanical unfolding of covalently linked GroES: Evidence of structural subunit intermediates

It is difficult to determine the structural stability of the individual subunits or protomers of many proteins in the cell that exist in an oligomeric or complexed state. In this study, we used single‐molecule force spectroscopy on seven subunits of covalently linked cochaperonin GroES (ESC7) to evaluate the structural stability of the subunit. A modified form of ESC7 was immobilized on a mica surface. The force‐extension profile obtained from the mechanical unfolding of this ESC7 showed a distinctive sawtooth pattern that is typical for multimodular proteins. When analyzed according to the worm‐like chain model, the contour lengths calculated from the peaks in the profile suggested that linked‐GroES subunits unfold in distinct steps after the oligomeric ring structure of ESC7 is disrupted. The evidence that structured subunits of ESC7 withstand external force to some extent even after the perturbation of the oligomeric ring structure suggests that a stable monomeric intermediate is an important component of the equilibrium unfolding reaction of GroES.     

Functional analysis of isolated cpn10 domains and conserved amino acid residues in spinach chloroplast co-chaperonin by site-directed mutagenesis

The possibilities of independent function of the two chaperonin 10 (cpn10) domains of the cpn10 homologue from spinach chloroplasts and the role of five conserved amino acid residues in the N-terminal cpn10 unit were investigated. Recombinant single domain proteins and complete chloroplast cpn10 proteins carrying amino acid exchanges of conserved residues in their N-terminal cpn10 domain were expressed in Escherichia coli and partially purified. The function of the recombinant proteins was tested using GroEL as chaperonin 60 (cpn60) partner for in vitro refolding of denatured ribulose-1,5-bisphosphate carboxylase (Rubisco). Interaction with cpn60 was also monitored by the ability to inhibit GroEL ATPase activity. In vitro both isolated cpn10 domains were found to be incapable of co-chaperonin function. All mutants were also severely impaired in cpn10 function. The results are interpreted in terms of an essential role of the exchanged amino acid residues for the interaction between co-chaperonin and cpn60 partner and in terms of a functional coupling of both cpn10 domains.To test the function of mutant chloroplast cpn10 proteins in vivo the cpn10 deficiency of E. coli strain CG712 resulting in an inability to assemble -phage was exploited in a complementation assay. Transformation with plasmids directing the expression of mutant chloroplast cpn10 proteins in two cases restored -phage assembly in this bacterial strain to the same extent as did transformation with a plasmid encoding wild-type cpn10 protein. In contrast a plasmid encoded third mutant and truncated forms of chloroplast cpn 10 showed significantly reduced complementation efficiencies.