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Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL.

ABSTRACT: In a newly isolated temperature-sensitive lethal Escherichia coli mutant affecting the chaperonin GroEL, we observed wholesale aggregation of newly translated proteins. After temperature shift, transcription, translation, and growth slowed over two to three generations, accompanied by filamentation and accretion (in approximately 2% of cells) of paracrystalline arrays containing mutant chaperonin complex. A biochemically isolated inclusion body fraction contained the collective of abundant proteins of the bacterial cytoplasm as determined by SDS/PAGE and proteolysis/MS analyses. Pulse-chase experiments revealed that newly made proteins, but not preexistent ones, were recruited to this insoluble fraction. Although aggregation of "stringent" GroEL/GroES-dependent substrates may secondarily produce an "avalanche" of aggregation, the observations raise the possibility, supported by in vitro refolding experiments, that the widespread aggregation reflects that GroEL function supports the proper folding of a majority of newly translated polypeptides, not just the limited number indicated by interaction studies and in vitro experiments.

SUBMITTER: Chapman E

PROVIDER: S-EPMC1613232 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Folding of newly translated membrane protein CCR5 is assisted by the chaperonin GroEL-GroES.

Project description:The in vitro folding of newly translated human CC chemokine receptor type 5 (CCR5), which belongs to the physiologically important family of G protein-coupled receptors (GPCRs), has been studied in a cell-free system supplemented with the surfactant Brij-35. The freshly synthesized CCR5 can spontaneously fold into its biologically active state but only slowly and inefficiently. However, on addition of the GroEL-GroES molecular chaperone system, the folding of the nascent CCR5 was significantly enhanced, as was the structural stability and functional expression of the soluble form of CCR5. The chaperonin GroEL was partially effective on its own, but for maximum efficiency both the GroEL and its GroES lid were necessary. These results are direct evidence for chaperone-assisted membrane protein folding and therefore demonstrate that GroEL-GroES may be implicated in the folding of membrane proteins.

| S-EPMC4653635 | biostudies-literature

The versatile mutational "repertoire" of Escherichia coli GroEL, a multidomain chaperonin nanomachine.

Project description:The bacterial chaperonins are highly sophisticated molecular nanomachines, controlled by the hydrolysis of ATP to dynamically trap and remove from the environment unstable protein molecules that are susceptible to denaturation and aggregation. Chaperonins also act to assist in the refolding of these unstable proteins, providing a means by which these proteins may return in active form to the complex environment of the cell. The Escherichia coli GroE chaperonin system is one of the largest protein supramolecular complexes known, whose quaternary structure is required for segregating aggregation-prone proteins. Over the course of more than two decades of research on GroE, it has become accepted that GroE, more specifically the GroEL subunit, is a "high-tolerance" molecular system, capable of accommodating numerous mutations, while retaining its molecular integrity. In some cases, a given site of mutation was revealed to be absolutely required for GroEL function, providing hints regarding the network of signals and triggers that propel this unique system. In other instances, however, a mutation has produced a more delicate response, altering only part of, or in some cases, only a single facet of, the molecular mechanism, and these mutants have often provided invaluable hints on the extent of the complexity underlying chaperonin-assisted protein folding. In this review, we highlight some examples of the latter type of GroEL mutants which compose the unique "mutational repertoire" of GroEL and touch upon the important clues that each mutant provided to the overall effort to elucidate the details of GroE action.

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In silico engineering of aggregation-prone recombinant proteins for substrate recognition by the chaperonin GroEL.

Project description:BACKGROUND: Molecular chaperones appear to have been evolved to facilitate protein folding in the cell through entrapment of folding intermediates on the interior of a large cavity formed between GroEL and its co-chaperonin GroES. They bind newly synthesized or non-native polypeptides through hydrophobic interactions and prevent their aggregation. Some proteins do not interact with GroEL, hence even though they are aggregation prone, cannot be assisted by GroEL for their folding. RESULTS: In this study, we have attempted to engineer these non-substrate proteins to convert them as the substrate for GroEL, without compromising on their function. We have used a computational biology approach to generate mutants of the selected proteins by selectively mutating residues in the hydrophobic patch, similar to GroES mobile loop region that are responsible for interaction with GroEL, and compared with the wild counterparts for calculation of their instability and aggregation propensities. The energies of the newly designed mutants were computed through molecular dynamics simulations. We observed increased aggregation propensity of some of the mutants formed after replacing charged amino acid residues with hydrophobic ones in the well defined hydrophobic patch, raising the possibility of their binding ability to GroEL. CONCLUSIONS: The newly generated mutants may provide potential substrates for Chaperonin GroEL, which can be experimentally generated and tested for their tendency of aggregation, interactions with GroEL and the possibility of chaperone-assisted folding to produce functional proteins.

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Chaperonin GroEL/GroES Over-Expression Promotes Aminoglycoside Resistance and Reduces Drug Susceptibilities in Escherichia coli Following Exposure to Sublethal Aminoglycoside Doses.

Project description:Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antibiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and over-expression sensitize and promote short-term tolerance, respectively, to this drug class. Here, we show that chaperonin GroEL/GroES over-expression accelerates acquisition of streptomycin resistance and reduces susceptibility to several other antibiotics following sub-lethal streptomycin antibiotic exposure. Chaperonin buffering could provide a novel mechanism for emergence of antibiotic resistance.

| S-EPMC4726795 | biostudies-literature

The Functional Differences between the GroEL Chaperonin of <i>Escherichia coli</i> and the HtpB Chaperonin of <i>Legionella pneumophila</i> Can Be Mapped to Specific Amino Acid Residues.

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Dataset concerning GroEL chaperonin interaction with proteins.

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| S-EPMC4735476 | biostudies-literature

Escherichia coli strain 8624 and Escherichia coli strain VS94 with signaling molecules

Project description:These E. coli strains were grown with various signaling molecules and the expression profiles were determined. Keywords: addition of quorum and host hormone signals

2007-09-20 | GSE7439 | GEO

Probing the dynamic process of encapsulation in Escherichia coli GroEL.

Project description:Kinetic analyses of GroE-assisted folding provide a dynamic sequence of molecular events that underlie chaperonin function. We used stopped-flow analysis of various fluorescent GroEL mutants to obtain details regarding the sequence of events that transpire immediately after ATP binding to GroEL and GroEL with prebound unfolded proteins. Characterization of GroEL CP86, a circularly permuted GroEL with the polypeptide ends relocated to the vicinity of the ATP binding site, showed that GroES binding and protection of unfolded protein from solution is achieved surprisingly early in the functional cycle, and in spite of greatly reduced apical domain movement. Analysis of fluorescent GroEL SR-1 and GroEL D398A variants suggested that among other factors, the presence of two GroEL rings and a specific conformational rearrangement of Helix M in GroEL contribute significantly to the rapid release of unfolded protein from the GroEL apical domain.

| S-EPMC3813556 | biostudies-literature

Creating the Functional Single-Ring GroEL-GroES Chaperonin Systems via Modulating GroEL-GroES Interaction.

Project description:Chaperonin and cochaperonin, represented by E. coli GroEL and GroES, are essential molecular chaperones for protein folding. The double-ring assembly of GroEL is required to function with GroES, and a single-ring GroEL variant GroELSR forms a stable complex with GroES, arresting the chaperoning reaction cycle. GroES I25 interacts with GroEL; however, mutations of I25 abolish GroES-GroEL interaction due to the seven-fold mutational amplification in heptameric GroES. To weaken GroELSR-GroES interaction in a controlled manner, we used groES 7, a gene linking seven copies of groES, to incorporate I25 mutations in selected GroES modules in GroES7. We generated GroES7 variants with different numbers of GroESI25A or GroESI25D modules and different arrangements of the mutated modules, and biochemically characterized their interactions with GroELSR. GroES7 variants with two mutated modules participated in GroELSR-mediated protein folding in vitro. GroES7 variants with two or three mutated modules collaborated with GroELSR to perform chaperone function in vivo: three GroES7 variants functioned with GroELSR under both normal and heat-shock conditions. Our studies on functional single-ring bacterial chaperonin systems are informative to the single-ring human mitochondrial chaperonin mtHsp60-mtHsp10, and will provide insights into how the double-ring bacterial system has evolved to the single-ring mtHsp60-mtHsp10.

| S-EPMC5575113 | biostudies-literature

Co-purification from Escherichia coli of a plant beta-glucosidase-glutathione S-transferase fusion protein and the bacterial chaperonin GroEL.

Project description:The coding sequence of the mature cyanogenic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) of Manihot esculenta Crantz (cassava) was cloned into the vector pGEX-2T and expressed in Escherichia coli. The bacterial chaperonin GroEL [Braig, Otwinowski, Hedge, Boisvert, Joachimiak, Horwich and Sigler (1994) Nature (London) 371, 578-586] was found to be tightly associated with the fusion protein and co-purified with it. In the presence of excess MgATP, release and folding of the fusion beta-glucosidase were demonstrated by a fast increase in both linamarase and p-nitrophenyl-beta-D-glucopyranosidase activity at a low protein concentration. A slow endogenous folding process was also detected by activity measurements. Michaelis constants (Km) and the ratio between the maximal velocities and efficiency constants (Vmax., Vmax./Km) for the hydrolysis of the natural substrate, linamarin, and p-nitrophenyl beta-D-glucopyranoside (PNP-Glc) by the recombinant protein were found to be almost identical with those of the native glycosylated plant enzyme [Keresztessy, Kiss and Hughes (1994) Arch. Biochem. Biophys. 314, 142-152]. Molecular dissociation constants for the free enzyme (pK(E)1, pK(E)2) obtained with linamarin and PNP-Glc, and the enzyme substrate complexes (pK(ES)1, pK(ES)2) were also in accordance with that of the original protein. The reactive substrate analogue N-bromoacetyl beta-D-glucosylamine inactivated the fusion enzyme according to pseudo-first-order kinetics with first-order rate constant (k1=0.007 min-1) and apparent inhibition constants (k1=20 mM) comparable with those of the plant protein [Keresztessy, Kiss and Hughes (1994) Arch. Biochem. Biophys. 315, 323-330]. In comparison with the native glycosylated plant protein, the recombinant protein was, however, found to be extremely sensitive to proteolysis and misfolding.

| S-EPMC1217050 | biostudies-other

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