Lysozyme reactivation using immobilized molecular chaperonin GroEL

The molecular chaperonin, GroEL, was immobilized to a porous matrix and used to reactivate denatured lysozyme. The maximum reactivation yield was obtained at 37°C and pH 6–8 and about 90% activity of the denatured lysozyme was restored under the conditions. The coupling density of GroEL had little effect on the chaperoning activity of GroEL up to 48 mg g^–1 gel. The immobilized GroEL was reusable, indicating the possibility of using it on a large scale for the refolding of proteins.     

Effect of hydrogen peroxide on the activity and structure of Escherichia coli chaperone GroEL

Chaperone GroEL was treated with different concentrations of hydrogen peroxide. The conformational states of GroEL were monitored by protein intrinsic fluorescence, 8-anilino-1-naphthalene sulfonate fluorescence, and far-UV CD measurements. The results show that GroEL has unusual ability to resist oxidative stress. GroEL kept its quaternary structure and activity even when treated with 10 mM hydrogen peroxide. Two fragments were formed when GroEL was treated with high concentrations of hydrogen peroxide (more than 20 mM). It is suggested that GroEL, as a molecular chaperone, is related to oxidative process in vivo.     

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。     

A structural model for the GroEL chaperonin

Abstract Individual particle analysis of end views from negatively stained specimensof purified GroEL from Escherichia coli showed the presence of two different particle populations, those with a six-fold symmetry and those with a seven-fold symmetry, when studied at pH 7.7 and 5.0. Image processing of particles from frozen-hydrated specimens revealed at both pH values a homogeneous population of particles with a strong seven-fold symmetry component and an average image with seven asymmetric units. Biochemical analysis of purified GroEL showed unequivocally the presence of a single polypeptide with the N-terminal sequence identical to that of GroEL. These results are compatible with a structural model of GroEL as an asymmetric aggregate built up by two rings of seven-fold and six-fold symmetries, respectively.     

Probing the "annealing" mechanism of GroEL minichaperone using molecular dynamics simulations

Although the intact chaperonin machinery is needed to rescue natural substrate proteins (SPs) under non-permissive conditions the "minichaperone" alone, containing only the isolated apical domain of GroEL, can assist folding of a certain class of proteins. To understand the annealing function of the minichaperone, we have carried out molecular dynamics simulations in the NPT ensemble totaling 300ns for four systems; namely, the isolated strongly binding peptide (SBP), the minichaperone, and the SBP and a weakly binding peptide (WBP) in complex with the minichaperone. The SBP, which is structureless in isolation, adopts a beta-hairpin conformation in complex with the minichaperone suggesting that favorable non-specific interactions of the SPs confined to helices H and I of the apical domains can induce local secondary structures. Comparison of the dynamical fluctuations of the apo and the liganded forms of the minichaperone shows that the stability (needed for SP capture) involves favorable hydrophobic interactions and hydrogen bond network formation between the SBP and WBP, and helices H and I. The release of the SP, which is required for the annealing action, involves water-mediated interactions of the charged residues at the ends of H and I helices. The simulation results are consistent with a transient binding release (TBR) model for the annealing action of the minichaperone. According to the TBR model, SP annealing occurs in two stages. In the first stage the SP is captured by the apical domain. This is followed by SP release (by thermal fluctuations) that places it in a different region of the energy landscape from which it can partition rapidly to the native state with probability Phi or be trapped in another misfolded state. The process of binding and release can result in enhancement of the native state yield. The TBR model suggests "that any cofactor that can repeatedly bind and release SPs can be effective in assisting protein folding." By comparing the structures of the non-chaperone alpha-casein (which has no sequence similarity with the apical domain) and the minichaperone and the hydrophobicity profiles we show that alpha-casein has a pair of helices that have similar sequence and structural profiles as H and I. Based on this comparison we identify residues that stabilize (destabilize) alpha-casein-protein complexes. This suggests that alpha-casein assists folding by the TBR mechanism.     

溶菌酶的研究进展

溶菌酶在自然界中广泛存在,是一种与单核-巨噬细胞系统有关的非特异性防御机制。人溶菌酶在临床上具有潜在的使用价值。由于来源有限,利用基因工程技术从细菌或酶母中生产人溶菌酶实现产业化,是解决其供需矛盾的有效途径。有关溶菌酶抗菌功能以外的其它未知生物学作用,也是当前研究的热点。     

Equine lysozyme: the molecular basis of folding, self-assembly and innate amyloid toxicity

Calcium-binding equine lysozyme (EL) combines the structural and folding properties of c-type lysozymes and alpha-lactalbumins, connecting these two most studied subfamilies. The structural insight into its native and partially folded states is particularly illuminating in revealing the general principles of protein folding, amyloid formation and its inhibition. Among lysozymes EL forms one of the most stable molten globules and shows the most uncooperative refolding kinetics. Its partially-folded states serve as precursors for calcium-dependent self-assembly into ring-shaped and linear amyloids. The innate amyloid cytotoxicity of the ubiquitous lysozyme highlights the universality of this phenomenon and necessitates stringent measures for its prevention.     

Kinetic characterization of rat liver nuclear lysozyme

The kinetic properties of rat liver nuclear lysozyme, earlier purified to homogeneity in our laboratory, have been studied. The enzyme was found to be maximally active in the pH range 4.2 to 5.4 in 0.02 M buffer. Its Km was found to be 333 mg/litre. It was heat sensitive even in the acidic pH range. The enzyme exhibited tissue specific differences when compared with the rat kidney nuclear lysozyme.     

Antibody response to GroEL varies in patients with acute Mycoplasma pneumoniae infection

Although heat-shock proteins represent major antigens in a wide spectrum of bacterial infections, their immunogenicity is not known for Mycoplasma pneumoniae. M. pneumoniae is a major human respiratory pathogen and it has been suggested that its groEL gene might be dispensable in vitro. Using the specific monoclonal antibody 2C2/C3 we found an abundant synthesis of about 58 kDa GroEL in M. pneumoniae reference strains and in 15 clinical isolates examined at low and higher passages. In patients with acute respiratory disease caused by M. pneumoniae immunoblot analyses showed relatively low prevalence of systemic antibodies against its GroEL protein. Whereas all patients had strong antibody response to the P1 adhesin, only 5 of 29 patients (17.2%) had antibodies to GroEL. Among them, patient RI raised an early and very strong antibody response to GroEL. During the convalescent phase, levels of his serum IgG (mainly IgG2) to GroEL increased and were higher than levels of IgG to P1.     

GroEL interacts transiently with oxidatively inactivated rhodanese facilitating its reactivation

When the enzyme rhodanese was inactivated with hydrogen peroxide (H(2)O(2)), it underwent significant conformational changes, leading to an increased exposure of hydrophobic surfaces. Thus, this protein seemed to be an ideal substrate for GroEL, since GroEL uses hydrophobic interactions to bind to its substrate polypeptides. Here, we report on the facilitated reactivation (86%) of H(2)O(2)-inactivated rhodanese by GroEL alone. Reactivation by GroEL required a reductant and the enzyme substrate, but not GroES or ATP. Further, we found that GroEL interacted weakly and/or transiently with H(2)O(2)-inactivated rhodanese. A strong interaction with rhodanese was obtained when the enzyme was pre-incubated with urea, indicating that exposure of hydrophobic surfaces alone on oxidized rhodanese was not sufficient for the formation of a strong complex and that a more unfolded structure of rhodanese was required to interact strongly with GroEL. Unlike prior studies that involved denaturation of rhodanese through chemical or thermal means, we have clearly shown that GroEL can function as a molecular chaperone in the reactivation of an oxidatively inactivated protein. Additionally, the mechanism for the GroEL-facilitated reactivation of rhodanese shown here appears to be different than that for the chaperonin-assisted folding of chemically unfolded polypeptides in which a nucleotide and sometimes GroES is required.     

GroEL/GroES chaperone and Lon protease regulate expression of the Vibrio fischeri lux operon in Escherichia coli

Chaperone GroEL/GroES and Lon protease were shown to play a role in regulating the expression of the Vibrio fischeri lux operon cloned in Escherichia coli cells. The E. coli groE mutant carrying a plasmid with the full-length V. fischeri lux regulon showed a decreased bioluminescence. The bioluminescence intensity was high in E. coli cells with mutant lonA and the same plasmid. Bioluminescence induction curves lacked the lag period characteristic of lon ⁺ strains. Regulatory luxR of V. fischeri was cloned in pGEX-KG to produce the hybrid gene GST-luxR. The product of its expression, GST-LuxR, was isolated together with GroEL and Lon upon affinity chromatography on a column with glutathione-agarose, suggesting complexation of LuxR with these proteins. It was assumed that GroEL/GroES is involved in LuxR folding, while Lon protease degrades LuxR before its folding into an active globule or after denaturation.     

Effect of ADP and GroES on interaction of molecular chaperonin GroEL with non-native lysozyme

The interaction of the molecular chaperonin GroEL with fluorescein-labeled lysozyme in the presence of high concentrations of thiol reagent--dithiothreitol (DTT) has been studied. In case of high concentrations of DTT lysozyme loses the native conformation due to the disruption of the intramolecular disulfide bonds stabilizing its structure and effectively aggregates. It has been shown that in the presence of high concentrations of DTT and two-fold molar excess of GroEL the lysozyme tightly interacts with GroEL that essentially decreases the efficiency of its aggregation. The addition of ADP to the complex of GroEL with nonnative lysozyme noticeably decreases the interaction of the chaperonin with nonnative protein target resulting in some increase of the efficiency of its aggregation. However, the addition of the co-chaperonin GroES together with ADP (i.e. the formation of the complex of GroEL with GroES) leads to drastic weakness of the interaction of GroEL with nonnative lysozyme and the efficiency of its aggregation becomes comparable with that in the absence of GroEL.     

Crystal structure of wild-type chaperonin GroEL

The 2.9A resolution crystal structure of apo wild-type GroEL was determined for the first time and represents the reference structure, facilitating the study of structural and functional differences observed in GroEL variants. Until now the crystal structure of the mutant Arg13Gly, Ala126Val GroEL was used for this purpose. We show that, due to the mutations as well as to the presence of a crystallographic symmetry, the ring-ring interface was inaccurately described. Analysis of the present structure allowed the definition of structural elements at this interface, essential for understanding the inter-ring allosteric signal transmission. We also show unambiguously that there is no ATP-induced 102 degrees rotation of the apical domain helix I around its helical axis, as previously assumed in the crystal structure of the (GroEL-KMgATP)(14) complex, and analyze the apical domain movements. These results enabled us to compare our structure with other GroEL crystal structures already published, allowing us to suggest a new route through which the allosteric signal for negative cooperativity propagates within the molecule. The proposed mechanism, supported by known mutagenesis data, underlines the importance of the switching of salt bridges.     

Localization of DnaK and GroEL in Vibrio cholerae

Though the GroEL and DnaK heat shock proteins are well characterized in prokaryotes, only scanty and controversial information exist about their cellular localization. In the present study, the localization of the heat shock proteins DnaK and GroEL in normal and heat shocked cells of Vibrio cholerae, was investigated both by immunogold labeling of ultrathin sections and biochemical methods. Much of the DnaK was found to be localized at the inner membrane in unstressed cells, most probably at the Bayer's adhesion sites. Data suggested that upon heat shock, the DnaK associated with the membrane continued to remain there, but the newly synthesized DnaK appeared mostly in the cytoplasm. GroEL in both stressed and unstressed cells was found mainly in the cytoplasm.     

溶菌酶的研究进展

溶菌酶普遍存在于动物、植物和微生物中。溶菌酶是一种小分子碱性蛋白,长期以来一直被作为一种“模型”体系．用于研究蛋白质的空间构象、酶动力学及其与分子进化、分子免疫间的关系。介绍了溶菌酶的来源、结构、性质、作用机制。并对近年来其在食品工业、医学和酶工程中的应用进行了综述;分析了溶菌酶应用中存在的主要问题,并对其应用前景进行了展望。     

Identification and characterization of a GroEL homologue in Rhodobacter sphaeroides

A protein closely related to the Escherichia coli GroEL protein has been isolated from Rhodobacter sphaeroides. Native and SDS-polyacrylamide gel electrophoresis of this protein have shown that it is present in the cell as a multimeric complex of Mr 670,000 which is composed of a monomer of Mr 58,000. Antisera raised against the Mr 58,000 polypeptide have been shown to cross-react with GroEL and the alpha subunit of the pea plastid chaperonin. The N-terminal amino acid sequence of the Mr 58,000 polypeptide is identical to that of GroEL at 15 of 19 residues and is also closely related to the alpha subunit of the pea plastid chaperonin, though less so to the beta subunit.     

Stability and release requirements of the complexes of GroEL with two homologous mammalian aminotransferases

The mitochondrial (mAAT) and cytosolic (cAAT) homologous isozymes of aspartate aminotransferase are two relatively large proteins that in their nonnative states interact very differently with GroEL. MgATP alone can increase the rate of GroEL-assisted reactivation of cAAT, yet the presence of GroES is mandatory for mAAT. Addition of an excess of a denatured substrate accelerates reactivation of cAAT in the presence of GroEL, but has no effect on mAAT. These competition studies suggest that the more stringent substrate mAAT forms a thermodynamically stable complex with GroEL, while rebinding affects the slow reactivation kinetics of cAAT with GroEL alone. However, the competitor appears to accelerate the release of cAAT from GroEL, most likely by displacing bound cAAT from the GroEL cavity. Moreover, cAAT, but not mAAT, shows a time-dependent increase in protease resistance while bound to GroEL at low temperature. These results suggest that folding and release of cAAT from GroEL in the absence of cofactors may occur stepwise with certain interactions being broken and reformed until the protein escapes binding. The distinct behavior of these two isozymes most likely results from differences in the structure of the nonnative states that bind to GroEL.     

Glycosylation inhibits the interaction of invertase with the chaperone GroEL.

During refolding and reassociation of chemically denatured non-glycosylated invertase from Saccharomyces cerevisiae, aggregation competes with correct folding, leading to low yields of reactivation (Kern et al. (1992) Protein Sci. 1, 120-131). In the presence of the chaperone GroEL, refolding is completely arrested. This suggests the formation of a stable complex between GroEL and non-native non-glycosylated invertase. Addition of MgATP results in a slow release of active invertase from the chaperone complex. When GroEL/ES and MgATP are present during refolding, the final reactivation yield increases from 14% to 36%. In contrast, refolding of the core-glycosylated and the high-mannose glycosylated forms of invertase is not arrested by GroEL. Only a short lag phase at the beginning of reactivation and a slightly increased reactivation yield (64% to 86% for core-glycosylated and 62% to 76% for external invertase) indicate a weak interaction of the glycosylated forms with the chaperone.     

大肠杆菌异源重组蛋白质的变性与复性

描述了大肠杆菌异源重组蛋白质的形成、制备、变性和复性,综述了国内外变性、复性的有效方法。     

Affinity chromatography of GroEL chaperonin based on denatured proteins: Role of electrostatic interactions in regulation of GroEL affinity for protein substrates

The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.