分子伴侣研究概况

分子伴侣主要是在进化上高度保守的热休克蛋白的几个家族。从细菌到哺乳动物,分子伴侣对体内蛋白质的折叠、运输和组装都起到非常重要的作用。本文简要地概述了分子伴侣的组成、它们在蛋白质折叠中的作用以及它们在生物工程下游处理过程中的应用情况。     

分子伴侣

分子伴侣是最近十几年才发现的一类非常保守的蛋白家庭。它与酶的作用方式类似，能和某些不同的多肽链非特异性结合，催化介导蛋白质特定构象的形成，参与体内蛋白质的折叠、装配和转运，但又不构成其结构的一部分。这类保守的蛋白家族大致可分为四类，广泛存在于生物体中。其中研究得最多的是热休克蛋白。实际上，分子伴侣是一种蛋白质分子构象的协助者，主要参与蛋白质次级结构的形成。     

保守GTP酶EF-G在蛋白质合成中的功能研究北大核心CSCD

延伸因子G(elongation factor G,EF-G)是一种保守的GTP水解酶,它是蛋白质翻译过程中一个重要的调控因子。同源模拟发现EF-G与核糖体保护蛋白Tet(O)具有相似的空间结构且都包含5个结构域,序列比对发现E.coliEF-G与Campylobacter jejuniTet(O)结构域Ⅳ保守的两个环状区不同。通过分子克隆构建EF-G嵌合体,表达纯化后的蛋白突变体通过核糖体依赖的GTP水解酶(GTPase)活性检测、多聚尿嘧啶(polyU)为mRNA合成苯丙氨酸多肽链、多聚核糖体的解聚检测及相关的体内实验,检测EF-G在肽链合成中的作用,结果发现EF-G嵌合体能够影响肽链生成过程中tRNA-mRNA复合物的移位,但不影响核糖体的再循环过程。     

玉米细胞质分子伴侣Hsp70的ATPase活性

玉米胚乳细胞中纯化的细胞质Ｈｓｐ７０蛋白有低水平的ＡＴＰａｓｅ 活性,它在５０ ℃、ｐＨ５ ．８ 、２０ ｍｍｏｌ／Ｌ的ＫＣｌ 条件下活性最高,Ｃａ２＋和Ｍｇ２＋ 抑制其活性。大肠杆菌ＤｎａＪ蛋白能将玉米细胞质Ｈｓｐ７０ 的ＡＴＰａｓｅ 活性提高６倍,而ＧｒｐＥ 蛋白对其影响很小。８ 种不同的人工合成多肽均能刺激该蛋白的ＡＴＰａｓｅ 活性,增加幅度从２ ．５ 倍到１０ 倍不等。亲水性不同的氨基酸对Ｈｓｐ７０ 的ＡＴＰａｓｅ 活性影响不同。玉米细胞质Ｈｓｐ７０ 是一个三磷酸核苷酸酶,除ＡＴＰ 外,它还能催化ＵＴＰ、ＧＴＰ、ＣＴＰ和ＩＴＰ的水解     

分子伴侣在蛋白质折叠中的作用

分子伴侣主要由三个高度保守的蛋白质家族组成,这三个家族的成员广泛分布于原核和真核细胞中。TCP1复合物是真核细胞细胞溶质内的伴侣蛋白。分子伴侣在蛋白质折叠过程中防止多肽链形成聚集物或无活性结构,提高正确折叠率。本文重点讨论Stress-70家族蛋白质和伴侣蛋白协助蛋白质折叠过程中的协同性以及伴侣蛋白GroEL和GroES的作用机理。     

应用分子伴侣共表达系统表达pfu基因及酶活性测定

将通过In-fution方法构建的pET32a-pfu质粒与可以促进可溶性表达的HG-PGR07质粒一起转入大肠杆菌B121（DE3）共表达,以pET32a-pfu单独在B121（DE3）中表达作为对照。用热变性和（NH4）2SO4沉淀去除部分杂蛋白,再经Ni—NAT亲和层析柱纯化分离尼血蛋白,SDS-PAGE检测结果表明目的蛋白大小约为90kD,与预计的分子量大小一致。最后对其酶活性测定结果表明分子伴侣能够促进pfu基因表达,并能够提高酶活性。     

病毒与分子伴侣

分子伴侣与病毒生命活动密切相关,从病毒复制的起始、转录的进行、翻译的完成到病毒粒子的装配成熟,甚至病毒在宿主体内的转运都有分子伴侣的参与。随着病毒与分子伴侣相互关系研究的深入,产生了抗病毒的又一可能途径。     

GroEL分子伴侣研究进展

大肠杆菌的GroEL是同型寡聚复合体,由14个相对分子质量58×103亚基组成背靠背的双环结构。它在新生蛋白质的正确折叠和组装以及在热或化学逆境下变性蛋白质的恢复过程中起重要作用。同时,在大肠杆菌的跨膜转运及插入细胞质膜方面都起重要作用。这些活动依赖于GroEL与底物蛋白的疏水片断的相互作用。综述了Hsp60分子伴侣系统中研究得比较清楚的GroEL的晶体结构、功能及作用机理等方面的研究进展。     

玉米细胞质分子伴侣Hsp70的ATPase活性

玉米胚乳细胞中纯化的细胞质Ｈｓｐ７０蛋白有低水平的ＡＴＰａｓｅ活性,它在５０℃、ＰＨ５．８、２０ｍｍｏｌ／Ｌ的ＫＣｌ条件下活性最高,Ｃａ＾２＋和Ｍｇ＾２＋抑制其活性。大肠杆菌ＤｎａＪ蛋白能将玉米细胞质Ｈｓｐ７０的ＡＴＰａｓｅ活性提高６倍,而ＧｒｐＥ蛋白对其影响很小。８种不同的人工合成多肽均能刺激该蛋白折ＡＴＰａｓｅ活性,增加幅度从２．５倍到１０倍痫水性不同的氨基酸对Ｈｓｐ７０的ＡＴＰａｓｅ活性影响     

HdeB Functions as an Acid-protective Chaperone in Bacteria

Enteric bacteria such as Escherichia coli utilize various acid response systems to counteract the acidic environment of the mammalian stomach. To protect their periplasmic proteome against rapid acid-mediated damage, bacteria contain the acid-activated periplasmic chaperones HdeA and HdeB. Activation of HdeA at pH 2 was shown to correlate with its acid-induced dissociation into partially unfolded monomers. In contrast, HdeB, which has high structural similarities to HdeA, shows negligible chaperone activity at pH 2 and only modest chaperone activity at pH 3. These results raised intriguing questions concerning the physiological role of HdeB in bacteria, its activation mechanism, and the structural requirements for its function as a molecular chaperone. In this study, we conducted structural and biochemical studies that revealed that HdeB indeed works as an effective molecular chaperone. However, in contrast to HdeA, whose chaperone function is optimal at pH 2, the chaperone function of HdeB is optimal at pH 4, at which HdeB is still fully dimeric and largely folded. NMR, analytical ultracentrifugation, and fluorescence studies suggest that the highly dynamic nature of HdeB at pH 4 alleviates the need for monomerization and partial unfolding. Once activated, HdeB binds various unfolding client proteins, prevents their aggregation, and supports their refolding upon subsequent neutralization. Overexpression of HdeA promotes bacterial survival at pH 2 and 3, whereas overexpression of HdeB positively affects bacterial growth at pH 4. These studies demonstrate how two structurally homologous proteins with seemingly identical in vivo functions have evolved to provide bacteria with the means for surviving a range of acidic protein-unfolding conditions.     

转谷氨酰胺酶基因在大肠杆菌中的克隆表达

从轮枝链霉菌Streptoverticilliummobaraense细胞中获得其基因组DNA ,用一对特异性的引物通过PCR的方法扩增出转谷氨酰胺酶 (transglutaminase,TGase)全长基因 ,回收片段并将其连接到表达载体pET30a中 ,转化大肠杆菌DH5α。双向测序表明获得的转谷氨酰胺酶全长基因序列正确。纯化重组质粒转化大肠杆菌BL2 1 (DE3) ,以 1mmol/LIPTG诱导 5h收集菌体进行SDS-PAGE电泳分析 ,与阴性对照相比 ,明显多出了一条蛋白条带 ,紫外扫描显示此带约占总蛋白量的1 7% ,Westernblotting证实此带能够特异性地与兔抗MTG(味之素公司 )的抗体发生反应。测得纯化后得到的TGase蛋白的酶活可以达到 15.1U/mg蛋白。     

Chaperone characteristics of PDI-related protein A from Aspergillus niger

The functional properties of a novel protein, protein disulfide isomerase-related protein A (PRPA) from Aspergillus niger T21, have been characterized. (1) PRPA possesses disulfide isomerase activity. (2) In Hepes buffer, at substoichiometric concentrations, PRPA facilitates the formation of inactive lysozyme aggregates associated with PRPA (anti-chaperone activity); while at a high molar excess, PRPA inhibits aggregation by maintaining lysozyme in a soluble, yet inactive, state (chaperone-like activity). However, PRPA only exhibits chaperone-like activity during lysozyme refolding in phosphate buffer. (3) Experiments have indicated that disulfide cross-linkage is not required for the interaction between PRPA and lysozyme, and hydrophobic interaction may be responsible for PRPA effect on lysozyme. (4) Co-expression of PRPA and prochymosin in Escherichia coli leads to reduction of inclusion bodies, rendering part of prochymosin molecules soluble yet inactive. The structural and functional characteristics of PRPA suggest that PRPA may play an important role in protein folding, aggregation, and retention in the endoplasmic reticulum.     

Conserved,Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide- and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.     

Dynamic Interaction of the Sec Translocon with the Chaperone PpiD

The Sec translocon constitutes a ubiquitous protein transport channel that consists in bacteria of the three core components: SecY, SecE, and SecG. Additional proteins interact with SecYEG during different stages of protein transport. During targeting, SecYEG interacts with SecA, the SRP receptor, or the ribosome. Protein transport into or across the membrane is then facilitated by the interaction of SecYEG with YidC and the SecDFYajC complex. During protein transport, SecYEG is likely to interact also with the protein quality control machinery, but details about this interaction are missing. By in vivo and in vitro site-directed cross-linking, we show here that the periplasmic chaperone PpiD is located in front of the lateral gate of SecY, through which transmembrane domains exit the SecY channel. The strongest contacts were found to helix 2b of SecY. Blue native PAGE analyses verify the presence of a SecYEG-PpiD complex in native Escherichia coli membranes. The PpiD-SecY interaction was not influenced by the addition of SecA and only weakly influenced by binding of nontranslating ribosomes to SecYEG. In contrast, PpiD lost contact to the lateral gate of SecY during membrane protein insertion. These data identify PpiD as an additional and transient subunit of the bacterial SecYEG translocon. The data furthermore demonstrate the highly modular and versatile composition of the Sec translocon, which is probably essential for its ability to transport a wide range of substrates across membranes in bacteria and eukaryotes.     

Modulation of the Chaperone DnaK Allosterism by the Nucleotide Exchange Factor GrpE

Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70_NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70_SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaK_NBD, with ATP inducing the open, substrate-receptive DnaK_SBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaK_NBD. DnaK_SBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaK_NBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaK_SBD near DnaK_NBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaK_SBD, whereas the DnaK_NBD-DnaK_SBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.