Comparison of Folding Rates of Homologous Prokaryotic and Eukaryotic Proteins

Cholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitro disassembly and reassembly of CtxB 5 (the B subunit pentamer of cholera toxin) is investigated. When CtxB 5 was acidified at pH 1.0 and then neutralized, the B-subunits disassembled and could no longer migrate as SDS-stable pentamers on polyacrylamide gels or be captured by GM1. However, continued incubation at neutral pH resulted in the B-subunits regaining the capacity to be detected by GM1 enzymelinked immunosorbent assay (t1 ⁄2 ϳ 8 min) and to migrate as SDS-stable pentamers (t1 ⁄2 ϳ 15 min). Time-dependent changes in Trp fluorescence intensity during B-subunit reassembly occurred with a half-time of ϳ8 min, similar to that detected by GM1 enzyme-linked immunosorbent assay, suggesting that both methods monitor earlier events than B-pentamer formation alone. Based on the Trp fluorescence intensity measurements, a kinetic model of the pathway of CtxB 5 reassembly was generated that depended on trans to cis isomerization of Pro-93 to give an interface capable of subunit-subunit interaction. The model suggests formation of intermediates in the reaction, and these were successfully detected by glutaraldehyde cross-linking.Cholera toxin (Ctx) 1 and heat-labile enterotoxin (Etx) are the primary virulence factors produced by Vibrio cholerae and certain toxinogenic strains of Escherichia coli, respectively (1-3). Both toxins are heterooligomeric proteins comprising an A-subunit that exhibits ADP-ribosyltransferase activity and five Bsubunits that bind with high affinity to the glycolipid receptor, monosialoganglioside GM1, found in the plasma membranes of mammalian cells (4 -6). The B pentamer components of both cholera toxin (CtxB 5 ) and E. coli heat-labile enterotoxin (EtxB 5 ) are widely thought of as carrier molecules principally involved in delivering the toxin A-subunit into cells (3). However, more recent studies have revealed that these receptor binding moieties possess striking immunomodulatory properties that can down-regulate inflammatory immune reactions (7-9). Such findings have prompted renewed interest in the B-subunit pentamers and led to their testing as a potential therapeutic agents for the treatment of inflammatory allergic and autoimmune disorders (10 -13).Assembly of Ctx and Etx into AB 5 complexes occurs in the periplasmic compartment of the bacterial cell envelope (14 -16). Expression of either CtxB or EtxB in the absence of their corresponding A-subunits results in the formation of highly stable B-subunit pentamers that are devoid of enterotoxic activity. The in vivo pathway of B-subunit pentamerization is poorly understood, chiefly because of the difficulty of investigating such processes in the complex environment of the periplasmic space (17, 18). The use of in vitro conditions to study the disassembly and reassembly of the toxins was first reported by Finkelstein et ...

“…Such a slow increase in fluorescence is unlikely to be monitoring a conformational change associated with the early stages of folding (for review see Refs. [27][28][29].…”

Section: Resultsmentioning

confidence: 99%

A Kinetic Model of Intermediate Formation during Assembly of Cholera Toxin B-subunit Pentamers

Lesieur¹,

Cliff²,

Carter³

et al. 2002

“…Unfolding-It has been reported that the rate of refolding of chicken cAAT and mAAT decreased by a factor of 4 and 25, respectively, when the proteins were denatured in 6 M GdnHCl for 40 s instead of for 40 min (29). These results were interpreted as indicating that proline isomerization is the ratelimiting step in the slow refolding of these enzymes unfolded for long periods of time.…”

Section: Reactivation Kinetics Of Plp-maat After Different Times Ofmentioning

confidence: 98%

“…These steps follow the rapid collapse of the unfolded chain into a molten globule-like intermediate. It has been proposed that isomerization of the two cis-prolines in AAT is responsible for the two slow phases observed during in vitro refolding of chicken mAAT (29) or E. coli AAT (30). However, analysis of the folding properties of P138A and P195A mutants of E. coli AAT indicated that although proline isomerization may play some role, other conformational rearrangements must rule the folding of this protein (31,32).…”

mentioning

confidence: 99%

The Nature of the Rate-limiting Steps in the Refolding of the Cofactor-dependent Protein Aspartate Aminotransferase

Oses-Prieto

Bengoechea-Alonso

Artigues

et al. 2003

The refolding of mitochondrial aspartate aminotransferase (mAAT; EC 2.6.1.1) has been studied following unfolding in 6 M guanidine hydrochloride for different periods of time. Whereas reactivation of equilibriumunfolded mAAT is sigmoidal, reactivation of the short term unfolded protein displays a double exponential behavior consistent with the presence of fast and slow refolding species. The amplitude of the fast phase decreases with increasing unfolding times (k Ϸ 0.75 min ؊1 at 20°C) and becomes undetectable at equilibrium unfolding. According to hydrogen exchange and stoppedflow intrinsic fluorescence data, unfolding of mAAT appears to be complete in less than 10 s, but hydrolysis of the Schiff base linking the coenzyme pyridoxal 5-phosphate (PLP) to the polypeptide is much slower (k Ϸ 0.08 min ؊1 ). This implies the existence in short term unfolded samples of unfolded species with PLP still attached. However, since the disappearance of the fast refolding phase is about 10-fold faster than the release of PLP, the fast refolding phase does not correspond to folding of the coenzyme-containing molecules. The fast refolding phase disappears more rapidly in the pyridoxamine and apoenzyme forms of mAAT, both of which lack covalently attached cofactor. Thus, bound PLP increases the kinetic stability of the fast refolding unfolding intermediates. Conversion between fast and slow folding forms also takes place in an early folding intermediate. The presence of cyclophilin has no effect on the reactivation of either equilibrium or short term unfolded mAAT. These results suggest that proline isomerization may not be the only factor determining the slow refolding of this cofactor-dependent protein.The folding and unfolding of proteins are complex processes affected by a number of features such as size, chain topology, and oligomeric state and ultimately by the amino acid sequence, which encodes each unique three-dimensional structure (1). Much of our understanding of the protein folding problem arises from in vitro refolding studies of small monomeric proteins (2). However, the molecular mass of the majority of proteins found in prokaryotic or eukaryotic cells is larger than 25 kDa (3). Most of these larger polypeptides fold into several structural domains, and in the case of oligomeric proteins several subunits associate through noncovalent interactions. Folding and unfolding of many large proteins with several folding domains and/or multiple subunits often show multiphasic kinetics with fast and slow folding phases, suggesting the presence of either multiple paths or intermediate states in the process (4). Slow folding phases are often associated with proline isomerization (5, 6), but in some cases the rate-limiting step involves, at least to some extent, the reorganization of misfolded species or metastable kinetic folding intermediates (7-9). The significance of these kinetic intermediates is still a matter of discussion. They are considered to represent either productive intermediates that facilitate folding or kinet...

“…Proline Isomerization and Folding of PmMDH-Comparative folding studies between homologous prokaryotic and eukaryotic aspartate transaminase performed by Widmann and Christen (14) indicate that the slower folding rate of the mitochondrial enzyme is caused, in part, by differences in proline isomerization (14). A sequence comparison between EcMDH and PmMDH reveals that there are more proline residues present within the mitochondrial primary sequence than in its bacterial homolog (21 versus 13 prolines, respectively).…”

Section: Refolding Reactions Of Ecmdh With Pmmdh At Physiological Temmentioning

confidence: 99%

“…In the absence of chaperonins, EcMDH has been found to reactivate much more rapidly than PmMDH at 25°C (14). In this study, we have examined the refolding of EcMDH at a physiological temperature of 37°C to determine whether Ec-MDH will now require chaperonins to aid in its folding.…”

mentioning

confidence: 99%

A Comparison of the GroE Chaperonin Requirements for Sequentially and Structurally Homologous Malate Dehydrogenases

Tieman¹,

Johnston

Fisher

2001

Escherichia coli malate dehydrogenase (EcMDH) and its eukaryotic counterpart, porcine mitochondrial malate dehydrogenase (PmMDH), are highly homologous proteins with significant sequence identity (60%) and virtually identical native structural folds. Despite this homology, EcMDH folds rapidly and efficiently in vitro and does not seem to interact with GroE chaperonins at physiological temperatures (37°C), whereas PmMDH folds much slower than EcMDH and requires these chaperonins to fold to the native state at 37°C. Double jump experiments indicate that the slow folding behavior of PmMDH is not limited by proline isomerization. Although the folding enhancer glycerol (<5 M) does not alter the renaturation kinetics of EcMDH, it dramatically accelerates the spontaneous renaturation of PmMDH at all temperatures tested. Kinetic analysis of PmMDH renaturation with increasing glycerol concentrations suggests that this osmolyte increases the onpathway kinetics of the monomer folding to assemblycompetent forms. Other osmolytes such as trimethylamine N-oxide, sucrose, and betaine also reactivate PmMDH at nonpermissive temperatures (37°C). Glycerol jump experiments with preformed GroEL⅐PmMDH complexes indicate that the shift between stringent (requires ATP and GroES) and relaxed (only requires ATP) complex conformations is rapid (<3-5 s). The similarity in irreversible misfolding kinetics of PmMDH measured with glycerol or the activated chaperonin complex (GroEL⅐GroES⅐ATP) suggests that these folding aids may influence the same step in the PmMDH folding reaction. Moreover, the interactions between glycerol-induced PmMDH folding intermediates and GroEL⅐GroES⅐ATP are diminished. Our results support the notion that the protein folding kinetics of sequentially and structurally homologous proteins, rather than the structural fold, dictates the GroE chaperonin requirement.GroEL is a complex, allosteric, protein folding machine whose function is controlled by associations with nucleotides, the co-chaperonin GroES, and substrate polypeptides (1-3). As with all allosteric proteins, ligand binding influences the structural constraints within the system, which in turn ultimately induce shifts between various functional states. Although detailed information is available on the structures of GroEL, with and without bound nucleotide, and of one GroEL⅐GroES complex, the exact mechanism(s) explaining chaperonin-assisted folding of substrate proteins remain(s) unclear. Differences in binding and conditions for productive release of substrate proteins are routinely observed, but the structural and energetic basis of these differences is not understood at the molecular level. Although molten globule folding intermediates have been suggested to be preferred substrates for chaperonins, the structures of these intermediate populations have broad distributions thus making it difficult to identify specific transient conformations that interact with the GroE chaperonins.In vitro and in vivo studies have shown that many proteins can interact with and...