Phosphocarrier proteins in an intracellular symbiotic bacterium of aphids.

A GroEL homolog produced by Buchnera, an intracellular symbiotic bacterium of aphids, is not only a molecular chaperone but also a novel phosphocarrier protein, suggesting that this protein plays a role in a signal transducing system specific to bacteria living in an intracellular environment. This prompted us to look into phosphocarrier proteins of Buchnera that may be shared in common with other bacteria. As a result, no evidence was obtained for the presence of sensor kinases of the two-component system in Buchnera, which are found in many bacteria. It is possible that the lack of sensor kinases is compensated for by the mulitifunctional GroEL homolog in this symbiotic bacteria. In contrast, we successfully identified three phosphotransferase system genes, ptsH, ptsI, and crr in Buchnera, and provide evidence for their active expression. While the deduced amino acid sequences of these gene products, histidine-containing phosphocarrier protein, Enzyme I, and Enzyme III were similar to their counterparts in Escherichia coli, the predicted isoelectric points of the Buchnera proteins were strikingly higher. It was also suggested that Buchnera Enzyme I, when produced in E. coli, is able to accept the phosphoryl group from phosphoenolpyruvate, but not from ATP.     

Provision of riboflavin to the host aphid,Acyrthosiphon pisum,by endosymbiotic bacteria,Buchnera

Differential cDNA display and quantitative RT-PCR suggested that the riboflavin synthase complex of the aphid endosymbiont, Buchnera, is active only when the symbiotic system is maintained and well organized in young hosts. Since this finding suggested the provision of riboflavin by Buchnera, we examined the effect of dietary riboflavin on the performance of symbiotic and aposymbiotic aphids using chemically-defined diets. Our results indicate: (1) dietary riboflavin is slightly detrimental to young, symbiotic aphids; (2) dietary riboflavin is essential to aposymbiotic aphids; (3) dietary riboflavin remarkably improves the performance of aposymbiotic aphids. These results strongly suggest that young, symbiotic aphids are provided with riboflavin by their endosymbionts, Buchnera.     

GroEL-mediated protein folding: making the impossible, possible

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins-constrained by sequence, topology, size, and function-simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.     

The N-terminal region of the luteovirus readthrough domain determines virus binding to Buchnera GroEL and is essential for virus persistence in the aphid.

Luteoviruses and the luteovirus-like pea enation mosaic virus (PEMV; genus Enamovirus) are transmitted by aphids in a circulative, nonreplicative manner. Acquired virus particles persist for several weeks in the aphid hemolymph, in which a GroEL homolog, produced by the primary endosymbiont of the aphid, is abundantly present. Six subgroup II luteoviruses and PEMV displayed a specific but differential affinity for Escherichia coli GroEL and GroEL homologs isolated from the endosymbiotic bacteria of both vector and nonvector aphid species. These observations suggest that the basic virus-binding capacity resides in a conserved region of the GroEL molecule, although other GroEL domains may influence the efficiency of binding. Purified luteovirus and enamovirus particles contain a major 22-kDa coat protein (CP) and lesser amounts of an approximately 54-kDa readthrough protein, expressed by translational readthrough of the CP into the adjacent open reading frame. Beet western yellows luteovirus (BWYV) mutants devoid of the readthrough domain (RTD) did not bind to Buchnera GroEL, demonstrating that the RTD (and not the highly conserved CP) contains the determinants for GroEL binding. In vivo studies showed that virions of these BWYV mutants were significantly less persistent in the aphid hemolymph than were virions containing the readthrough protein. These data suggest that the Buchnera GroEL-RTD interaction protects the virus from rapid degradation in the aphid. Sequence comparison analysis of the RTDs of different luteoviruses and PEMV identified conserved residues potentially important in the interaction with Buchnera GroEL.     

GroEL-Mediated Protein Folding: Making the Impossible,Possible

ABSTRACT

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins—constrained by sequence, topology, size, and function—simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.     

Triggering protein folding within the GroEL-GroES complex

The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.     

A newly synthesized protein interacts with GroES on the surface of chaperonin GroEL.

To facilitate folding and assembly of different proteins, chaperonin GroEL requires the presence of its helper protein GroES. Using a photochemical cross-linking approach, we show that GroES and newly synthesized pre-beta-lactamase (pre-beta lac) contact with each other only within the ternary complex with GroEL. Possibly owing to this contact GroES is able to directly influence the pre-beta lac/GroEL interaction. Furthermore, the cross-linking of pre-beta lac to GroES suggests that the binding of the protein ligands to GroEL occurs near the GroES binding site, known to be in the central hole space of GroEL.     

Analysis of peptides and proteins in their binding to GroEL

The GroEL–GroES is an essential molecular chaperon system that assists protein folding in cell. Binding of various substrate proteins to GroEL is one of the key aspects in GroEL‐assisted protein folding. Small peptides may mimic segments of the substrate proteins in contact with GroEL and allow detailed structural analysis of the interactions. A model peptide SBP has been shown to bind to a region in GroEL that is important for binding of substrate proteins. Here, we investigated whether the observed GroEL–SBP interaction represented those of GroEL–substrate proteins, and whether SBP was able to mimic various aspects of substrate proteins in GroE‐assisted protein folding cycle. We found that SBP competed with substrate proteins, including α‐lactalbumin, rhodanese, and malate dehydrogenase, in binding to GroEL. SBP stimulated GroEL ATP hydrolysis rate in a manner similar to that of α‐lactalbumin. SBP did not prevent GroES from binding to GroEL, and GroES association reduced the ATPase rates of GroEL/SBP and GroEL/α‐lactalbumin to a comparable extent. Binding of both SBP and α‐lactalbumin to apo GroEL was dominated by hydrophobic interaction. Interestingly, association of α‐lactalbumin to GroEL/GroES was thermodynamically distinct from that to GroEL with reduced affinity and decreased contribution from hydrophobic interaction. However, SBP did not display such differential binding behaviors to apo GroEL and GroEL/GroES, likely due to the lack of a contiguous polypeptide chain that links all of the bound peptide fragments. Nevertheless, studies using peptides provide valuable information on the nature of GroEL–substrate protein interaction, which is central to understand the mechanism of GroEL‐assisted protein folding. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Adaptive evolution in GroEL from distantly related endosymbiotic bacteria of insects

Many symbioses between bacteria and insects resulted from ancient infections followed by strict vertical transmission within host lineages. The strong bottlenecks under which this transmission occurs promote the neutral fixation of slightly deleterious mutations by genetic drift. As predicted by Muller's ratchet, this fixation will drive endosymbiotic bacteria through an irreversible dynamics of fitness loss. The chaperonin GroEL has been proposed as a compensatory mechanism whereby endosymbiotic bacteria of aphids persist. Here, we show that endosymbiotic bacteria of insects from two phylogenetically very distant bacterial phyla have fixed amino acid substitutions by positive selection in functionally important GroEL regions involved in either GroES/peptide binding or in the en bloc movement of the GroEL apical domain. These results, together with the high levels of constitutive expression of GroEL in these endosymbionts, provide valuable insights into the evolution of a molecular mechanism responsible for the maintenance of the symbiotic lifestyle.     

Mapping pathways of allosteric communication in GroEL by analysis of correlated mutations

An interesting example of an allosteric protein is the chaperonin GroEL. It undergoes adenosine 5'-triphosphate-induced conformational changes that are reflected in binding of adenosine 5'-triphosphate with positive cooperativity within rings and negative cooperativity between rings. Herein, correlated mutations in chaperonins are analyzed to unravel routes of allosteric communication in GroEL and in its complex with its co-chaperonin GroES. It is shown that analysis of correlated mutations in the chaperonin family can provide information about pathways of allosteric communication within GroEL and between GroEL and GroES. The results are discussed in the context of available structural, genetic, and biochemical data concerning short- and long-range interactions in the GroE system.     

A genomic reappraisal of symbiotic function in the aphid/Buchnera symbiosis: reduced transporter sets and variable membrane organisations

Buchnera aphidicola is an obligate symbiotic bacterium that sustains the physiology of aphids by complementing their exclusive phloem sap diet. In this study, we reappraised the transport function of different Buchnera strains, from the aphids Acyrthosiphon pisum, Schizaphis graminum, Baizongia pistaciae and Cinara cedri, using the re-annotation of their transmembrane proteins coupled with an exploration of their metabolic networks. Although metabolic analyses revealed high interdependencies between the host and the bacteria, we demonstrate here that transport in Buchnera is assured by low transporter diversity, when compared to free-living bacteria, being mostly based on a few general transporters, some of which probably have lost their substrate specificity. Moreover, in the four strains studied, an astonishing lack of inner-membrane importers was observed. In Buchnera, the transport function has been shaped by the distinct selective constraints occurring in the Aphididae lineages. Buchnera from A. pisum and S. graminum have a three-membraned system and similar sets of transporters corresponding to most compound classes. Transmission electronic microscopic observations and confocal microscopic analysis of intracellular pH fields revealed that Buchnera does not show any of the typical structures and properties observed in integrated organelles. Buchnera from B. pistaciae seem to possess a unique double membrane system and has, accordingly, lost all of its outer-membrane integral proteins. Lastly, Buchnera from C. cedri revealed an extremely poor repertoire of transporters, with almost no ATP-driven active transport left, despite the clear persistence of the ancestral three-membraned system.     

伴侣分子（GroE）纯化及其催化的重组蛋白质折叠反应

我们自Ｅ．ｃｏｌｉ细胞中纯化出ＧｒｏＥＬ和ＧｒｏＥＳ,对其有活性的分子状态和反应条件进行了探索,结果表明,只有在等摩尔的ＧｒｏＥＬ和ＧｒｏＥＳ以及１ｍｍｏｌ／ＬＡＴＰ和适当浓度的Ｋ＋存在时;才会有较高的催化折叠效率,它可使ｌｍｇ／ｍｌ的ＩＬ－２的正确折叠率由３０％提高到５８％,使ＩＬ－２和ＧＭ－ＣＳＦ的比活性提高１倍以上。它提高重组蛋白质正确拆叠率的关键是可以降低折叠过程中形成聚合体。     

Multiple structural transitions of the GroEL subunit are sensitive to intermolecular interactions with cochaperonin and refolding polypeptide

In this study we attempted to determine the specific roles of the numerous conformational changes that are observed in the bacterial chaperonin GroEL, by performing stopped-flow experiments on GroEL R231W in the presence of a refolding substrate protein. The apparent rate of one kinetic phase was decreased by approximately 25% in the presence of prebound unfolded malate dehydrogenase while another phase was suppressed completely under the same conditions, reflecting different effects of the unfolded protein on multiple structural transitions within GroEL. The addition of cochaperonin GroES counteracts the effect of the bound substrate protein in the former case, but had no effect on the latter, more extensive suppression. Using a chemically modified form of GroEL R231W which is incapable of releasing substrate proteins at low temperatures, we identified a conformational transition that is implicated in the release of substrate proteins. Parts of the actual process of substrate protein release were also observed through fluorescence resonance energy transfer experiments involving GroEL and labeled substrate protein. Analysis of the energy transfer data revealed an interesting relationship between substrate protein displacement and a specific structural transition in the GroEL apical domain.     

Chaperone activity of a chimeric GroEL protein that can exist in a single or double ring form.

The molecular chaperone GroEL is a protein complex consisting of two rings each of seven identical subunits. It is thought to act by providing a cavity in which a protein substrate can fold into a form that has no propensity to aggregate. Substrate proteins are sequestered in the cavity while they fold, and prevented from diffusion out of the cavity by the action of the GroES complex, that caps the open end of the cavity. A key step in the mechanism of action of GroEL is the transmission of a conformational change between the two rings, induced by the binding of nucleotides to the GroEL ring opposite to the one containing the polypeptide substrate. This conformational change then leads to the discharge of GroES from GroEL, enabling polypeptide release. Single ring forms of GroEL are thus predicted to be unable to chaperone the folding of GroES-dependent substrates efficiently, since they are unable to discharge the bound GroES and unable to release folded protein. We describe here a detailed functional analysis of a chimeric GroEL protein, which we show to exist in solution in equilibrium between single and double ring forms. We demonstrate that whereas the double ring form of the GroEL chimera functions effectively in refolding of a GroES-dependent substrate, the single ring form does not. The single ring form of the chimera, however, is able to chaperone the folding of a substrate that does not require GroES for its efficient folding. We further demonstrate that the double ring structure of GroEL is likely to be required for its activity in vivo.     

Functional characterization of an archaeal GroEL/GroES chaperonin system: significance of substrate encapsulation

In all three kingdoms of life chaperonins assist the folding of a range of newly synthesized proteins. As shown recently, Archaea of the genus Methanosarcina contain both group I (GroEL/GroES) and group II (thermosome) chaperonins in the cytosol. Here we report on a detailed functional analysis of the archaeal GroEL/GroES system of Methanosarcina mazei (Mm) in comparison to its bacterial counterpart from Escherichia coli (Ec). We find that the groESgroEL operon of M. mazei is unable to functionally replace groESgroEL in E. coli. However, the MmGroES protein can largely complement a mutant EcGroES protein in vivo. The ATPase rate of MmGroEL is very low and the dissociation of MmGroES from MmGroEL is 15 times slower than for the EcGroEL/GroES system. This slow ATPase cycle results in a prolonged enclosure time for model substrate proteins, such as rhodanese, in the MmGroEL:GroES folding cage before their release into the medium. Interestingly, optimal functionality of MmGroEL/GroES and its ability to encapsulate larger proteins, such as malate dehydrogenase, requires the presence of ammonium sulfate in vitro. In the absence of ammonium sulfate, malate dehydrogenase fails to be encapsulated by GroES and rather cycles on and off the GroEL trans ring in a non-productive reaction. These results indicate that the archaeal GroEL/GroES system has preserved the basic encapsulation mechanism of bacterial GroEL and suggest that it has adjusted the length of its reaction cycle to the slower growth rates of Archaea. Additionally, the release of only the folded protein from the GroEL/GroES cage may prevent adverse interactions of the GroEL substrates with the thermosome, which is not normally located within the same compartment.     

Mimicking the action of GroEL in molecular dynamics simulations: application to the refinement of protein structures

Bacterial chaperonin, GroEL, together with its co-chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL. GroES then encapsulates the substrate and triggers its release into the central cavity of the GroEL/ES complex for folding. In this work, we investigate the possibility to facilitate protein folding in molecular dynamics simulations by mimicking the effects of GroEL/ES namely, repeated binding and release, together with spatial confinement. During the binding stage, the (metastable) partially folded proteins are allowed to attach spontaneously to a hydrophobic surface within the simulation box. This destabilizes the structures, which are then transferred into a spatially confined cavity for folding. The approach has been tested by attempting to refine protein structural models generated using the ROSETTA procedure for ab initio structure prediction. Dramatic improvements in regard to the deviation of protein models from the corresponding experimental structures were observed. The results suggest that the primary effects of the GroEL/ES system can be mimicked in a simple coarse-grained manner and be used to facilitate protein folding in molecular dynamics simulations. Furthermore, the results support the assumption that the spatial confinement in GroEL/ES assists the folding of encapsulated proteins.     

Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity.

The mechanism of GroEL (chaperonin)-mediated protein folding is only partially understood. We have analysed structural and functional properties of the interaction between GroEL and the co-chaperonin GroES. The stoichiometry of the GroEL 14mer and the GroES 7mer in the functional holo-chaperonin is 1:1. GroES protects half of the GroEL subunits from proteolytic truncation of the approximately 50 C-terminal residues. Removal of this region results in an inhibition of the GroEL ATPase, mimicking the effect of GroES on full-length GroEL. Image analysis of electron micrographs revealed that GroES binding triggers conspicuous conformational changes both in the GroES adjacent end and at the opposite end of the GroEL cylinder. This apparently prohibits the association of a second GroES oligomer. Addition of denatured polypeptide leads to the appearance of irregularly shaped, stain-excluding masses within the GroEL double-ring, which are larger with bound alcohol oxidase (75 kDa) than with rhodanese (35 kDa). We conclude that the functional complex of GroEL and GroES is characterized by asymmetrical binding of GroES to one end of the GroEL cylinder and suggest that binding of the substrate protein occurs within the central cavity of GroEL.     

Stimulating the substrate folding activity of a single ring GroEL variant by modulating the cochaperonin GroES

In mediating protein folding, chaperonin GroEL and cochaperonin GroES form an enclosed chamber for substrate proteins in an ATP-dependent manner. The essential role of the double ring assembly of GroEL is demonstrated by the functional deficiency of the single ring GroEL(SR). The GroEL(SR)-GroES is highly stable with minimal ATPase activity. To restore the ATP cycle and the turnover of the folding chamber, we sought to weaken the GroEL(SR)-GroES interaction systematically by concatenating seven copies of groES to generate groES(7). GroES Ile-25, Val-26, and Leu-27, residues on the GroEL-GroES interface, were substituted with Asp on different groES modules of groES(7). GroES(7) variants activate ATP activity of GroEL(SR), but only some restore the substrate folding function of GroEL(SR), indicating a direct role of GroES in facilitating substrate folding through its dynamics with GroEL. Active GroEL(SR)-GroES(7) systems may resemble mammalian mitochondrial chaperonin systems.     

Action of the Chaperonin GroEL/ES on a Non-native Substrate Observed with Single-Molecule FRET

The double ring-shaped chaperonin GroEL binds a wide range of non-native polypeptides within its central cavity and, together with its cofactor GroES, assists their folding in an ATP-dependent manner. The conformational cycle of GroEL/ES has been studied extensively but little is known about how the environment in the central cavity affects substrate conformation. Here, we use the von Hippel-Lindau tumor suppressor protein VHL as a model substrate for studying the action of the GroEL/ES system on a bound polypeptide. Fluorescent labeling of pairs of sites on VHL for fluorescence (Förster) resonant energy transfer (FRET) allows VHL to be used to explore how GroEL binding and GroEL/ES/nucleotide binding affect the substrate conformation. On average, upon binding to GroEL, all pairs of labeling sites experience compaction relative to the unfolded protein while single-molecule FRET distributions show significant heterogeneity. Upon addition of GroES and ATP to close the GroEL cavity, on average further FRET increases occur between the two hydrophobic regions of VHL, accompanied by FRET decreases between the N- and C-termini. This suggests that ATP- and GroES-induced confinement within the GroEL cavity remodels bound polypeptides by causing expansion (or racking) of some regions and compaction of others, most notably, the hydrophobic core. However, single-molecule observations of the specific FRET changes for individual proteins at the moment of ATP/GroES addition reveal that a large fraction of the population shows the opposite behavior; that is, FRET decreases between the hydrophobic regions and FRET increases for the N- and C-termini. Our time-resolved single-molecule analysis reveals the underlying heterogeneity of the action of GroES/EL on a bound polypeptide substrate, which might arise from the random nature of the specific binding to the various identical subunits of GroEL, and might help explain why multiple rounds of binding and hydrolysis are required for some chaperonin substrates.     

GroEL-substrate-GroES ternary complexes are an important transient intermediate of the chaperonin cycle

GroEL C138W is a mutant form of Escherichia coli GroEL, which forms an arrested ternary complex composed of GroEL, the co-chaperonin GroES and the refolding protein molecule rhodanese at 25 degrees C. This state of arrest could be reversed with a simple increase in temperature. In this study, we found that GroEL C138W formed both stable trans- and cis-ternary complexes with a number of refolding proteins in addition to bovine rhodanese. These complexes could be reactivated by a temperature shift to obtain active refolded protein. The simultaneous binding of GroES and substrate to the cis ring suggested that an efficient transfer of substrate protein into the GroEL central cavity was assured by the binding of GroES prior to complete substrate release from the apical domain. Stopped-flow fluorescence spectroscopy of the mutant chaperonin revealed a temperature-dependent conformational change in GroEL C138W that acts as a trigger for complete protein release. The behavior of GroEL C138W was reflected closely in its in vivo characteristics, demonstrating the importance of this conformational change to the overall activity of GroEL.