GroEL‐Mediated protein folding

Fenton, Wayne A.; Horwich, Arthur L.

doi:10.1002/pro.5560060401

Cited by 330 publications

(231 citation statements)

References 124 publications

(144 reference statements)

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“…In the crowded cellular environment, protein folding is often aided by molecular chaperones. Molecular chaperones differ in size, function and energy dependence; however, all have in common to bind to the unfolded state of the protein in order to facilitate or mediate assembly of the correct three-dimensional structure 2,3 . In contrast to molecular chaperones, the intramolecular chaperones (IMCs) constitute a different class of chaperones.…”

Section: A R T I C L E Smentioning

confidence: 99%

Crystal structure of an intramolecular chaperone mediating triple–β-helix folding

Schulz

Dickmanns

Urlaub

et al. 2010

Nat Struct Mol Biol

106

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Proteins fold into their functional three-dimensional structure based on the information encoded in the residue sequence 1 . In all domains of life, various strategies evolved to assist folding processes in order to prevent immediate misfolding and aggregation of the nascent polypeptide chain. In the crowded cellular environment, protein folding is often aided by molecular chaperones. Molecular chaperones differ in size, function and energy dependence; however, all have in common to bind to the unfolded state of the protein in order to facilitate or mediate assembly of the correct three-dimensional structure 2,3 . In contrast to molecular chaperones, the intramolecular chaperones (IMCs) constitute a different class of chaperones. As part of the polypeptide chain, the IMC is typically cleaved off the target protein after the folding process is completed. Two classes of IMCs can be distinguished: class I IMCs assist the protein to fold into the correct tertiary structure, whereas class II IMCs are involved in quaternary structure assembly 4 . In contrast to many molecular chaperones, no evidence for an ATP-driven cleavage reaction could be found in IMCs. An example of a class II intramolecular chaperone has been identified in viral tailspike and fiber proteins. These proteins are functionally unrelated but share a highly conserved chaperone domain at their C terminus (C-terminal intramolecular chaperone domain, CIMCD), which is cleaved at a conserved position in an autoproteolytic reaction 5 . It was shown that the covalent linkage between the CIMCD and N-terminal pre-protein is necessary for correct folding, indicating that an in trans function of the chaperone is impossible 5 . Furthermore, it could be shown that some of the chaperone domains are exchangeable between the different preproteins 5,6 . While the three-dimensional structures of the CIMCDs are as yet unknown, the crystal structure of N-terminally truncated mature endoNF shows that the homotrimeric enzyme comprises a triple β-helix involved in substrate recognition 7 . This triple β-helix and the related triple-β-spiral motif have been identified in various proteins, which often play a role as virulence factors 8 . In triple β-helices, three polypeptide chains wind around a common threefold symmetry axis, conferring an extraordinary stability to the protein. The rigid elongated shape allows triple β-helix-comprising proteins to protrude from a pathogen's surface in order to interact with flexible host cell receptors, like lipopolysaccharides 9 . However, proper assembly of triple-β-helical folds poses to be difficult in the absence of a trimerization domain 10 . Hence, most triple β-helices depend on a C-terminal extension for trimerization and correct assembly 11 .Here we present the crystal structures of two representatives of a large group of systematically, functionally and structurally similar intramolecular chaperones: the Escherichia coli phage K1F endosilidase CIMCD and the Bacillus subtilis phage GA-1 neck appendage protein CIMCD. Furthermore...

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Section: A R T I C L E Smentioning

confidence: 99%

Crystal structure of an intramolecular chaperone mediating triple–β-helix folding

Schulz

Dickmanns

Urlaub

et al. 2010

Nat Struct Mol Biol

106

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“…This large binding site can bind to and accommodate very large protein domains. 17,40 Because of the tight binding interactions sometimes approach antibody antigen binding affinities, 41 hydrophobic proteins remain bound to the immobilized GroEL in the absence of ATP. Fortuitously, the double heptamer barrel shape of the biotinylated GroEL will invariably expose at least one if not both of its binding sites since this large barrel structure (14 3 14 nm 2 ) is presumably randomly orientated on the streptavidin BLI tip.…”

Section: Discussionmentioning

confidence: 99%

“…15,16 In this work, we demonstrate the analytical development of a novel chaperonin-based, biolayer interferometry (GroEL-BLI) assay system that has the potential to enable pharmaceutical scientists to detect structurally altered and/or early aggregate species of virtually any therapeutic protein candidate that transiently exposes hydrophobic surfaces. We used several pharmaceutically relevant proteins as models to demonstrate the ability of this GroEL-BLI biosensor methodology to rapidly detect preaggregate/aggregate formation in protein solutions during slightly elevated (40)(41)(42)(43)(44)(45) C) to moderate (55 C) thermal stress.…”

Section: Introductionmentioning

confidence: 99%

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio‐layer interferometry

et al. 2014

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The ability of a GroEL-based bio-layer interferometry (BLI) assay to detect structurally altered and/or aggregated species of pharmaceutically relevant proteins is demonstrated. Assay development included optimizing biotinylated-GroEL immobilization to streptavidin biosensors, combined with biophysical and activity measurements showing native and biotinylated GroEL are both stable and active. First, acidic fibroblast growth factor (FGF-1) was incubated under conditions known to promote (40 C) and inhibit (heparin addition) molten globule formation. Heat exposed (40 C) FGF-1 exhibited binding to GroEL-biosensors, which was significantly diminished in the presence of heparin. Second, a polyclonal human IgG solution containing 6-8% non-native dimer showed an increase in higher molecular weight aggregates upon heating by size exclusion chromatography (SEC). The poly IgG solution displayed binding to GroEL-biosensors initially with progressively increased binding upon heating. Enriched preparations of the IgG dimers or monomers showed significant binding to GroEL-biosensors. Finally, a thermally treated IgG1 monoclonal antibody (mAb) solution also demonstrated increased GroEL-biosensor binding, but with different Additional Supporting Information may be found in the online version of this article. Subhashchandra Naik and Ozan S. Kumru contributed equally to the work. kinetics. The bound complexes could be partially to fully dissociated after ATP addition (i.e., specific GroEL binding) depending on the protein, environmental stress, and the assay's experimental conditions. Transmission electron microscopy (TEM) images of GroEL-mAb complexes, released from the biosensor, also confirmed interaction of bound complexes at the GroEL binding site with heat-stressed mAb. Results indicate that the GroEL-biosensor-BLI method can detect conformationally altered and/or early aggregation states of proteins, and may potentially be useful as a rapid, stability-indicating biosensor assay for monitoring the structural integrity and physical stability of therapeutic protein candidates.

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“…The intermediate domain of GroEL links apical domain to equatorial domain and flanked by hinge region. This hinge region allows the movement of polypeptide in response to ATP and GroES binding [2][3][4]. The GroEL and GroES proteins are essential for bacteriophage λ growth in E. coli.…”

Section: Introductionmentioning

confidence: 99%