分子伴侣HdeA与底物蛋白SurA作用机制的模拟研究

分子伴侣HdeA与底物蛋白间的相互作用可帮助底物蛋白复性,这是肠道致病菌得以在酸性环境中幸存的重要原因之一.为探究HdeA发挥伴侣活性的作用机制,本研究采用分子对接和分子动力学的方法,模拟了HdeA与底物蛋白SurA间的相互作用,计算了二者的结合自由能.通过分析HdeA-SurA复合物体系的作用模式、氢键作用以及能量分解的结果,确定了HdeA与底物蛋白SurA结合时发挥重要作用的关键氨基酸残基.该研究结果为以后采用实验手段探究HdeA与底物蛋白之间的作用提供了重要的理论参考,同时为今后设计与开发HdeA的抑制剂提供了理论指导依据.     

保守GTP酶ObgE的分子伴侣活性研究

分子伴侣(molecular chaperone)能够帮助新生多肽链或错误折叠的蛋白质形成天然构象,但本身又不是成熟蛋白质的组成成分。蛋白质需要分子伴侣的帮助,才能够从核糖体合成的新生肽链折叠成有生物活性的大分子。E.coli的ObgE蛋白是保守的GTP酶,ObgE蛋白参与信号转导、蛋白运输和细胞周期调控,并与E.coli在氨基酸饥饿下的应激反应有关。本实验通过分子克隆,将E.coli ObgE蛋白的基因克隆到表达载体pET-28a中,转化到E.coli BL21进行蛋白表达纯化。纯化后的ObgE蛋白通过柠檬酸合成酶变复性实验、α-葡萄糖苷酶变复性实验、牛碳酸酐酶变复性实验,检测ObgE蛋白的分子伴侣活性,发现ObgE具有一定的分子伴侣活性,为该蛋白的研究应用奠定了基础。     

大肠杆菌分子伴侣GroE系统及其协助的Rubisco蛋白装配

分子伴侣协助蛋白质在体内正确组装,对分子伴侣结构和作用机制的研究不仅在生物大分子结构和功能研究中具有重要的理论意义,而且还具有广泛的应用价值。大肠杆菌分子伴侣GroE系统是迄今为止研究得最为透彻的分子伴侣。本文侧重总结了GroE系统的作用机制以及在该系统的帮助下光合细菌核酮糖1,5二磷酸羧化/加氧酶(Rubisco蛋白)的装配情况。     

分子伴侣过量表达对蛋白质分泌及可溶性的影响

 通过过量表达大肠杆菌分子伴侣 Sec B和 Gro EL,研究了它们对靶蛋白的分泌及可溶性的影响 .在过量表达 Sec B的宿主菌中 ,周质空间分泌蛋白总量较对照组提高了约 71 % ,GL- 7- ACA酰化酶在周质空间酶的活力较对照组提高了约 1 .5倍 ,碱性磷酸酯酶在周质空间酶的活力较对照组提高了约 54% ;在过量表达 Gro EL的宿主菌中 ,周质分泌蛋白总量较对照组提高了约 52 % ,青霉素 G酰化酶在周质空间酶的活力较对照组提高了约 76% ,鲑鱼降钙素六聚体的可溶性组分的比例由原来的 45%增加到约 90 % ,而 MS2 -人白介素 - 3融合蛋白的包涵体有约 1 5%转变为可溶性组份 .上述结果表明 ,分子伴侣 Sec B和 Gro EL的过量表达促进了靶蛋白的分泌 ,Gro EL增加了靶蛋白的可溶性     

分子伴侣及其在蛋白质折叠中的作用研究进展

蛋白质折叠是一个复杂的、动态的过程,蛋白质的折叠不是自发的,需要其他物质的帮助.了解分子伴侣在蛋白质折叠过程中的的作用,有助于进一步研究蛋白质折叠机制.本文介绍了分子伴侣及其分类,重点综述了各类分子伴侣在蛋白质折叠中的机制,并提出了研究分子伴侣在蛋白质折叠中的作用的重要意义.     

HSP70分子伴侣系统研究进展

综述了HSP70分子伴侣系统的晶体结构、功能及作用机理方面的研究进展.HSP70分子伴侣能够帮助细胞内新生蛋白的折叠和跨膜运输、蛋白质多聚体结构的装配和解装配,并能在胁迫下维持蛋白质的特殊构象,防止未折叠的蛋白质变性和使聚集的蛋白质溶解复性.所有这些活性均依赖于ATP调节的HSP70与底物蛋白中的疏水片段的相互作用.     

硫矿硫化叶菌P2分子伴侣基因的克隆表达及其性质

[目的]研究重组表达的硫矿硫化叶菌P2分子伴侣β亚基体外同源聚合体的结构和生化功能.[方法]利用PCR技术从硫矿硫化叶菌P2的基因组DNA中克隆得到分子伴侣β亚基的基因,将该基因克隆到表达载体pET-21a( )上并在大肠杆菌BL21(DE3)中实现了表达.对纯化后的β亚基单体进行体外聚合,利用透射电镜观察β分子伴侣的结构,并对其促蛋白折叠性质进行了研究.[结果]硫矿硫化叶菌P2分子伴侣β亚基基因在大肠杆菌BL21中实现了高效表达,纯化后的分子伴侣β亚基单体在ATP和Mg2 存在的条件下可自组装形成分子伴侣聚合体.透射电镜观察表明:该β分子伴侣具有Ⅱ型分子伴侣典型的双层面包圈结构,每个环由8个亚基构成.该β分子伴侣具有ATPase活性,最适反应温度为80℃;它不仅能够促进变性的绿色荧光蛋白(GFP)重新折叠,而且还能有效的提高木聚糖酶的热稳定性.[结论]本文根据P2基因组序列分析预测的分子伴侣基因设计引物,克隆表达了硫矿硫化叶菌P2分子伴侣的β亚基,纯化后对其进行体外聚合,透射电镜观察表明该聚合体具有Ⅱ型分子伴侣的经典结构,功能分析表明该β分子伴侣能够在体外促进异源蛋白质的折叠、提高其它酶分子的热稳定性.这为进一步深入研究嗜热古菌耐热抗逆的分子机制,奠定了良好的基础.     

分子内分子伴侣--Pro肽在蛋白质折叠中的作用

在体内,许多蛋白质,如很多胞外蛋白酶、某些多肽激素等都以含前导肽的前体形式合成,前导肽在蛋白质折叠中具有分子伴侣的功能。为了与一般意义上的分子伴侣相区别,人们将对蛋白质折叠有帮助的前导肽称为分子内分子伴侣,分子内分子伴侣帮助蛋白质在折叠过程中克服高的能量障碍,某些蛋白质的分子内分子伴侣甚至促进其在氧化性折叠中二硫键的正确配对。     

大肠杆菌分子伴侣GroF系统及其胁助的Rubisco蛋白装配

分子伴吕协助蛋白质在体内正确组装,对分子伴侣结构和作用机制的研究不仅在生物大分子结构和功能研究中具有重要的理论意义,而且还具有广泛应用价值。大肠杆菌分子伴侣ＧｒｏＥ系统是迄今为止研究得最为透彻的分子伴侣。本文侧重总结了ＧｒｏＥ系统的作用机制及在该系统的帮助下光合细菌核酮糖１,５－二磷酸羧化／加氧酶（Ｒｕｂｉｓｃｏ）的装配情况。     

大肠杆菌抗酸碱环境的分子机制

细胞抗酸碱机制是一个重要的分子生物学问题,也是当前国际上非常活跃的研究前沿领域。大肠杆菌的抗酸系统中,与代谢相关的抗酸机制是利用氨基酸脱羧酶消耗氨基酸特定的氢离子,产生胺类和CO2,最终提高pH值。周质空间的抗酸机制,主要是依靠分子伴侣来保护周质空间中蛋白质。细菌二元调控系统可以感知极端pH环境等外界刺激,激活或抑制靶基因的表达,使细胞做出应答。非编码小RNA的抗酸途径则通过增加RpoS或者降低H NS的翻译来实现。关于大肠杆菌抗碱机制的研究甚少,至今只发现gadC、rpoS、nhaA和tnaA四个基因可能帮助大肠杆菌细胞耐受极碱环境。揭示大肠杆菌抗酸碱途径的生物学机制,对人肠道致病菌的治疗和预防都有着积极的作用,也能为生物医药的研究提供可靠的分子依据和新药开发的新靶点。     

Escherichia coli HdeB is an acid stress chaperone

We cloned, expressed, and purified the hdeB gene product, which belongs to the hdeAB acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ion-exchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The hdeA and hdeB mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that Escherichia coli possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.     

Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.     

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.     

Salt bridges regulate both dimer formation and monomeric flexibility in HdeB and may have a role in periplasmic chaperone function

Escherichia coli and Gram-negative bacteria that live in the human gut must be able to tolerate rapid and large changes in environmental pH. Low pH irreversibly denatures and precipitates many bacterial proteins. While cytoplasmic proteins are well buffered against such swings, periplasmic proteins are not. Instead, it appears that some bacteria utilize chaperone proteins that stabilize periplasmic proteins, preventing their precipitation. Two highly expressed and related proteins, HdeA and HdeB, have been identified as acid-activated chaperones. The structure of HdeA is known and a mechanism for activation has been proposed. In this model, dimeric HdeA dissociates at low pH, and the exposed dimeric interface binds exposed hydrophobic surfaces of acid-denatured proteins, preventing their irreversible aggregation. We now report the structure and biophysical characterization of the HdeB protein. The monomer of HdeB shares a similar structure with HdeA, but its dimeric interface is different in composition and spatial location. We have used fluorescence to study the behavior of HdeB as pH is lowered, and like HdeA, it dissociates to monomers. We have identified one of the key intersubunit interactions that controls pH-induced monomerization. Our analysis identifies a structural interaction within the HdeB monomer that is disrupted as pH is lowered, leading to enhanced structural flexibility.     

Chaperone-dependent mechanisms for acid resistance in enteric bacteria

The extremely acidic environment of the mammalian stomach not only serves to facilitate food digestion but also acts as a natural barrier against infections of food-borne pathogens. Many pathogenic bacteria, such as enterohemorrhagic Escherichia coli, can breach this host defense and cause severe diseases. These pathogens have evolved multiple intricate strategies to overcome the bactericidal activity of acids. In particular, recent studies have uncovered the central roles of two periplasmic chaperones, HdeA and HdeB, in protecting enteric bacteria from extremely acidic conditions. Here, we review recent advances in the understanding of the acid resistance mechanisms of Gram-negative bacteria and focus on the mechanisms of HdeA and HdeB in preventing acid-induced protein aggregation and facilitating protein refolding following pH neutralization.     

A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance

Acid chaperones are essential factors in preserving the protein homeostasis for enteric pathogens to survive in the extremely acidic mammalian stomach (pH 1-3). The client proteins of these chaperones remain largely unknown, primarily because of the exceeding difficulty of determining protein-protein interactions under low-pH conditions. We developed a genetically encoded, highly efficient protein photocrosslinking probe, which enabled us to profile the in vivo substrates of a major acid-protection chaperone, HdeA, in Escherichia coli periplasm. Among the identified HdeA client proteins, the periplasmic chaperones DegP and SurA were initially found to be protected by HdeA at a low pH, but they subsequently facilitated the HdeA-mediated acid recovery of other client proteins. This unique, ATP-independent chaperone cooperation in the ATP-deprived E. coli periplasm may support the acid resistance of enteric bacteria. The crosslinker would be valuable in unveiling the physiological interaction partners of any given protein and thus their functions under normal and stress conditions.     

Evolutionary silence of the acid chaperone protein HdeB in enterohemorrhagic Escherichia coli O157:H7

The periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH < 3) in Escherichia coli and Shigella spp. Here we investigated the roles of HdeA and HdeB in the survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, the acid protections conferred by HdeA and HdeB in EHEC O145 were significant: loss of HdeA and HdeB led to over 100- to 1,000-fold reductions in acid survival, depending on the growth condition of prechallenge cells. However, this protection was much less in E. coli O157:H7 strains. Deletion of hdeB did not affect the acid survival of cells, and deletion of hdeA led to less than a 5-fold decrease in survival. Sequence analysis of the hdeAB operon revealed a point mutation at the putative start codon of the hdeB gene in all 26 E. coli O157:H7 strains analyzed, which shifted the ATG start codon to ATA. This mutation correlated with the lack of HdeB in E. coli O157:H7; however, the plasmid-borne O157-hdeB was able to restore partially the acid resistance in an E. coli O145ΔhdeAB mutant, suggesting the potential function of O157-HdeB as an acid chaperone. We conclude that E. coli O157:H7 strains have evolved acid survival strategies independent of the HdeA/B chaperones and are more acid resistant than nonpathogenic K-12 for cells grown under nonfavorable culturing conditions such as in Luria-Bertani no-salt broth at 28°C. These results suggest a divergent evolution of acid resistance mechanisms within E. coli.     

Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation

The extremely acidic environment of the mammalian stomach, with a pH range usually between 1 and 3, represents a stressful challenge for enteric pathogenic bacteria such as Escherichia coli before they enter into the intestine. The hdeA gene of E. coli was found to be acid inducible and was revealed by genetic studies to be important for the acid survival of the strain. This study was performed in an attempt to characterize the mechanism of the activity of the HdeA protein. Our data provided in this report strongly suggest that HdeA employs a novel strategy to modulate its chaperone activity: it possesses an ordered conformation that is unable to bind denatured substrate proteins under normal physiological conditions (i.e. at neutral pH) and transforms into a globally disordered conformation that is able to bind substrate proteins under stress conditions (i.e. at a pH below 3). Furthermore, our data indicate that HdeA exposes hydrophobic surfaces that appear to be involved in the binding of denatured substrate proteins at extremely low pH values. In light of our observations, models are proposed to explain the action of HdeA in both a physiological and a molecular context.     

Conserved amphiphilic feature is essential for periplasmic chaperone HdeA to support acid resistance in enteric bacteria

The extremely acidic environment of the mammalian stomach (pH 1-3) represents a stressful challenge for enteric pathogenic bacteria, including Escherichia coli, Shigella and Brucella. The hdeA (hns-dependent expression A) gene was found to be crucial for the survival of these enteric bacteria under extremely low pH conditions. We recently demonstrated that HdeA is able to exhibit chaperone-like activity exclusively within the stomach pH range by transforming from a well-folded conformation at higher pH values (above pH 3) into an unfolded conformation at extremely low pH values (below pH 3). This study was performed to characterize the action mechanisms and underlying specific structural features for HdeA to function in this unfolded conformation. In the present study, we demonstrate that the conserved 'amphiphilic' feature of HdeA, i.e. the exposure of the conserved hydrophobic region and highly charged terminal regions, is essential for exhibiting chaperone-like activity under extremely low pH conditions. Mutations that disrupt this amphiphilic feature markedly reduced the chaperone-like activity of HdeA. The results also strongly suggest that this acid-induced chaperone-like activity of HdeA is crucial for acid resistance of the enteric bacteria. Moreover, our new understanding of this amphiphilic structural feature of HdeA helps to better interpret how this unfolded (disordered) conformation could be functionally active.     

The Mechanism of HdeA Unfolding and Chaperone Activation

HdeA is a periplasmic chaperone that is rapidly activated upon shifting the pH to acidic conditions. This activation is thought to involve monomerization of HdeA. There is evidence that monomerization and partial unfolding allow the chaperone to bind to proteins denatured by low pH, thereby protecting them from aggregation. We analyzed the acid-induced unfolding of HdeA using NMR spectroscopy and fluorescence measurements, and obtained experimental evidence suggesting a complex mechanism in HdeA's acid-induced unfolding pathway, as previously postulated from molecular dynamics simulations. Counterintuitively, dissociation constant measurements show a stabilization of the HdeA dimer upon exposure to mildly acidic conditions. We provide experimental evidence that protonation of Glu37, a glutamate residue embedded in a hydrophobic pocket of HdeA, is important in controlling HdeA stabilization and thus the acid activation of this chaperone. Our data also reveal a sharp transition from folded dimer to unfolded monomer between pH 3 and pH 2, and suggest the existence of a low-populated, partially folded intermediate that could assist in chaperone activation or function. Overall, this study provides a detailed experimental investigation into the mechanism by which HdeA unfolds and activates.