Invitro effect of Canavalia ensiformis urease and the derived peptide Jaburetox-2Ec on Rhodnius prolixus Malpighian tubules

Ureases are metalloenzymes that are widespread among plants, fungi and bacteria. Urease isoforms (jack bean urease-JBU and canatoxin) from Canavalia ensiformis seeds are toxic to insects and fungi, suggesting a role in plant defense. The entomotoxic effect is due to the release of a 10-kDa peptide by cathepsin-like enzymes in the insect's midgut. Urease causes a decrease in post-feeding weight loss in Rhodnius prolixus, suggesting an effect on water balance. To investigate how this impairment occurs, we have evaluated the action of JBU and the urease-derivated peptide Jaburetox-2Ec on R. prolixus Malpighian tubules and also investigated the involvement of second messengers. JBU and Jaburetox-2Ec affect serotonin-induced secretion from Malpighian tubules. This effect is not cAMP-dependent, but the Jaburetox-2Ec effect is cGMP-dependent. Eicosanoid metabolites and calcium ions appear to be involved in JBU effect on diuresis, but are not involved in the action of Jaburetox-2Ec. Jaburetox-2Ec, but not JBU, causes a change in the transepithelial potential of the tubules. Canatoxin has a similar effect on tubules secretion, decreasing the secretion rate, but the urease from Helicobacter pylori has no significant effect. These data are helpful in our understanding of the actions of ureases and derived peptides on insects, and also reinforces the potential use of these proteins as biopesticides.     

Natriuresis and diuretic hormone synergism in R. prolixus upper Malpighian tubules is inhibited by the anti-diuretic hormone, RhoprCAPA-α2

Insects contain an array of hormones that coordinate the actions of the excretory system to achieve osmotic and ionic balance. In the hematophagous insect, Rhodnius prolixus, two diuretic hormones have been identified, serotonin (5HT) and a corticotropin releasing factor-related peptide (RhoprDH), and both lead to an increase in fluid secretion by Malpighian tubules (MTs). However, only 5HT activates reabsorption by the lower MTs to recover K(+) and Cl(-). An anti-diuretic hormone (RhoprCAPA-α2) is believed to coordinate the cessation of the rapid diuresis following blood meal engorgement. However, the role of RhoprCAPA-α2 on fluid secretion by MTs stimulated by RhoprDH was previously unknown. Here we demonstrate that, unlike the inhibitory effect on 5HT-stimulated secretion by MTs, RhoprCAPA-α2 does not inhibit secretion stimulated by RhoprDH although it does abolish the synergism that occurs between the two diuretic hormones. In addition, we show that the natriuresis elicited by either diuretic hormone is reduced by RhoprCAPA-α2. Using electrophysiological tools, we investigate the possible mechanism by which this complex regulatory pathway is achieved. Analysis of the pH of secreted fluid as well as the triphasic response in transepithelial potential in MTs treated with diuretic hormones, suggests that RhoprCAPA-α2 does not inhibit the V-type H(+) ATPase. Taken together, these results indicate that RhoprCAPA-α2 functions to reduce the rapid diuresis following blood feeding, and in addition, it inhibits the natriuresis associated with diuretic hormone stimulated MTs. This may reflect an important regulatory mechanism related to the slow diuresis that occurs as the K(+)-rich blood cells are digested.     

Jack bean urease alters serotonin-induced effects on Rhodnius prolixus anterior midgut

Urease isoforms from jack bean seeds are toxic to insects, and this entomotoxic effect is mostly due to the release of a peptide by insect digestive enzymes. We previously demonstrated that jack bean urease (JBU) has antidiuretic effects on Rhodnius prolixus Malpighian tubules, decreasing the serotonin-stimulated secretion of fluid. Now, we evaluate the toxicity of the intact JBU and its effect on R. prolixus anterior midgut, to further elucidate the mechanism of action of JBU in insects. JBU decreases the serotonin-induced fluid transport by the anterior midgut in vitro when injected into the lumen. A decrease in the levels of cAMP is observed in tissues treated with JBU (in the presence of serotonin). JBU also causes a dose-dependent increase in the frequency of serotonin-induced contractions in the anterior midgut, but does not alter the frequency of spontaneous contractions. The cyclooxygenase inhibitor indomethacin and the prostaglandin antagonist AH6809 block JBU's potentiation of serotonin-induced contractions, indicating that prostaglandins might act as second messengers for JBU action. Prostaglandin E₂ (PGE₂) increases the frequency of serotonin-induced contractions, again supporting the role of prostaglandins as second messengers for JBU action. JBU and PGE₂ increase cGMP levels in the anterior midgut, indicating that this molecule might also be part of the JBU pathway.     

Jaburetox-2Ec: an insecticidal peptide derived from an isoform of urease from the plant Canavalia ensiformis

Canatoxin, a urease isoform from Canavalia ensiformis seeds, shows insecticidal activity against different insect species. Its toxicity relies on an internal 10 kDa peptide (pepcanatox), released by hydrolysis of Canatoxin by cathepsins in the digestive system of susceptible insects. In the present work, based on the N-terminal sequence of pepcanatox, we have designed primers to amplify by PCR a 270-bp fragment corresponding to pepcanatox using JBURE-II cDNA (one of the urease isoforms cloned from C. ensiformis, with high identity to JBURE-I, the classical urease) as a template. This amplicon named jaburetox-2 was cloned into pET 101 vector to obtain heterologous expression in Escherichia coli of the recombinant protein in C-terminal fusion with V-5 epitope and 6-His tag. Jaburetox-2Ec was purified on Nickel-NTA resin and bioassayed in insect models. Dysdercus peruvianus larvae were fed on cotton seed meal diets containing 0.01% (w/w) Jaburetox-2Ec and, after 11 days, all individuals were dead. Jaburetox-2Ec was also tested against Spodoptera frugiperda larvae and caused 100% mortality. In contrast, high doses of Jaburetox-2Ec were innocuous when injected or ingested by mice and neonate rats. Modeling of Jaburetox-2Ec, in comparison with other peptide structures, revealed a prominent beta-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or cell permeation.     

Global and targeted proteomics in developing jack bean (Canavalia ensiformis) seedlings: an investigation of urease isoforms mobilization in early stages of development

Jack bean (Canavalia ensiformis) seeds are toxic for insects and the toxicity is due in part to an entomotoxic peptide enzymatically released from ureases in the midgut of susceptible insects. To characterize expression of urease isoforms in jack bean seed, particularly the more abundant urease isoform (JBU), quantitative proteomics was performed. Quiescent through 5-day germinating seeds were analyzed at 1-day intervals using a total proteomics approach (TPA) and also after co-immunoprecipitation (co-IP) with anti-JBU monoclonal antibodies. Jack bean proteins for TPA and co-IP were pre-fractionated by SDS-PAGE, segmented for in-gel trypsin digestion, and analyzed by liquid chromatography coupled to nanospray ionization tandem mass spectrometry (LC-MS/MS). Acquired MS(2) data were searched against a comprehensive plant database and the MEROPS peptidase database, in the absence of a jack bean EST database. Proteins detected in TPA were quantified by label-free spectral counting. A total of 234 and 106 non-redundant proteins were detected in TPA and co-IP, respectively. Mobilization of JBU was observed beginning 3-days after imbibition indicating that the entomotoxic peptide was not formed before this stage. A predicted urease isoform, JBURE-IIb, was detected in the co-IP study. Additionally, 46 plastid proteins, including RuBisCO and plastid ATPase were pulled down with JBU antibodies. These data shed new light on the behavior of urease isoforms during the early stages of plant development.     

Effect of chemical modification of histidines on the copper-induced oligomerization of jack bean urease (EC 3.5.1.5)

Aggregation of jack bean urease (JBU) is associated with alterations of its biological properties, notably the ureolytic and entomotoxic activities. We investigated the influence of metals on protein oligomerization and biological properties. Besides protein aggregation, Cu(2+) induces inhibition of both ureolytic and insecticidal activities of JBU. Chemical modification of histidine residues in JBU with diethylpyrocarbonate (DEPC) decreases its affinity for Cu(2+) and inhibits oligomerization induced by this metal. Furthermore, this modification protects the insecticidal properties of JBU from being inactivated by Cu(2+). Although DEPC-treated JBU displayed lower ureolytic activity, the modified protein is less susceptible to inhibition by Cu(2+) when compared to native enzyme. Our findings show that Cu(2+) promotes JBU aggregation and differently of other heavy metals studied here, it apparently inhibits the ureolytic activity by inducing protein polymerization along with blockage of sulfhydryl groups.     

Inhibition of the SAPK/JNK pathway blocks the stimulatory effects of glutamine on fluid secretion by the Malpighian tubules of Rhodnius prolixus

Physiological levels of amino acids such as glutamine, glutamate, aspartate and proline increase the rates of fluid secretion and ion transport by serotonin-stimulated Malpighian tubules (MTs) of Rhodnius prolixus. Here, we examine the proposal that the effects of glutamine are mediated through activation of specific kinases to produce the observed increases in fluid secretion. The glutamine-dependent increase in MT fluid secretion rate was blocked by two chemically unrelated inhibitors of the stress activated protein kinase (SAPK) pathway, SP600125 and dicumarol. Inhibitors of phosphatidyl inositol-3 kinase, p38 mitogen activated protein kinase (MAPK), extracellular-signal regulated kinases and MAPK kinase did not block glutamine's effects on fluid secretion rate when applied at commonly used concentrations. Inhibitors of protein kinase A or C reduced fluid secretion rates of serotonin-stimulated MTs, but did not block the response to glutamine. The glutamine-dependent increase in fluid secretion was also insensitive to cytoskeletal disrupting agents and protein synthesis inhibitors. Results of this study are the first to suggest a role for the SAPK pathway in the control of fluid secretion rates by insect MTs.     

Membrane-disruptive properties of the bioinsecticide Jaburetox-2Ec: Implications to the mechanism of the action of insecticidal peptides derived from ureases

Jaburetox-2Ec, a recombinant peptide derived from an urease isoform (JBURE-II), displays high insecticidal activity against important pests such as Spodoptera frugiperda and Dysdercus peruvianus. Although the molecular mechanism of action of ureases-derived peptides remains unclear, previous ab initio data suggest the presence of structural motifs in Jaburetox-2Ec with characteristics similar to those found in a class of pore-forming peptides. Here, we investigated the molecular aspects of the interaction between Jaburetox-2Ec and large unilamellar vesicles. Jaburetox-2Ec displays membrane-disruptive ability on acidic lipid bilayers and this effect is greatly influenced by peptide aggregation. Corroborating with this finding, molecular modeling studies revealed that Jaburetox-2Ec might adopt a well-defined β-hairpin conformation similar to those found in antimicrobial peptides with membrane disruption properties. In addition, molecular dynamics simulations suggest that the protein is able to anchor at a polar/non-polar interface. In the light of these findings, for the first time it was possible to point out some evidence that the peptide Jaburetox-2Ec interacting with lipid vesicles promotes membrane permeabilization.     

Urea metabolism in isolated perfused lungs of the laboratory rat and of a desert rodent, Notomys alexis

1. Using the isolated perfused lung preparation we have demonstrated a low-activity ureolytic enzyme present in rodent lung tissue. The enzyme shares four characteristic features with jack bean urease (EC 3.5.1.5). 2. Ureolytic activity was inhibited by fluoride ions and methionine hydroxamic acid; using the latter inhibitor, the I50 value and maximum inhibition were similar to those reported for jack bean urease. The apparent Km for rat lung urease was similar to the plasma urea level. 3. The low level of urease activity in the rat lung and in that of Notomys alexis, a desert rodent, suggests that the enzyme is not involved in urea excretion, rather that pulmonary ammonia production may influence fluid balance at the alveolus.     

Crystal Structure of the First Plant Urease from Jack Bean: 83 Years of Journey from Its First Crystal to Molecular Structure

Urease, a nickel-dependent metalloenzyme, is synthesized by plants, some bacteria, and fungi. It catalyzes the hydrolysis of urea into ammonia and carbon dioxide. Although the amino acid sequences of plant and bacterial ureases are closely related, some biological activities differ significantly. Plant ureases but not bacterial ureases possess insecticidal properties independent of its ureolytic activity. To date, the structural information is available only for bacterial ureases although the jack bean urease (Canavalia ensiformis; JBU), the best-studied plant urease, was the first enzyme to be crystallized in 1926. To better understand the biological properties of plant ureases including the mechanism of insecticidal activity, we initiated the structural studies on some of them. Here, we report the crystal structure of JBU, the first plant urease structure, at 2.05 Å resolution. The active-site architecture of JBU is similar to that of bacterial ureases containing a bi-nickel center. JBU has a bound phosphate and covalently modified residue (Cys592) by β-mercaptoethanol at its active site, and the concomitant binding of multiple inhibitors (phosphate and β-mercaptoethanol) is not observed so far in bacterial ureases. By correlating the structural information of JBU with the available biophysical and biochemical data on insecticidal properties of plant ureases, we hypothesize that the amphipathic β-hairpin located in the entomotoxic peptide region of plant ureases might form a membrane insertion β-barrel as found in β-pore-forming toxins.     

Jackbean, soybean and Bacillus pasteurii ureases: biological effects unrelated to ureolytic activity.

In this work we compared two plant ureases, jackbean urease (JBU) and embryo-specific soybean urease (SBU) and a bacterial (Bacillus pasteurii) urease, for kinetic parameters and other biological properties described recently for ureases that are independent of the ureolytic activity. The insecticidal effect of ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Contrasting with B. pasteurii urease (PBU), both plant ureases presented potent insecticidal activity, with LD(50) values of 0.017% (w/w) and 0.052% (w/w) for JBU and SBU, respectively. The insecticidal property of JBU or SBU was not affected by treatment with p-hydroxymercuribenzoate, an irreversible inhibitor of ureolytic activity of both proteins. Also, contrasting with canatoxin - a urease isoform from jackbean seeds that displays a toxic effect in mice (LD(50) = 2 mg x kg(-1)) - no lethality was seen in mice injected intraperitoneally with JBU or SBU (20 mg x kg(-1)). Similarly to canatoxin, the three enzymes promoted aggregation of blood platelets (EC(50) = 400.0 micro g x mL(-1), 22.2 micro g x mL(-1), 15.8 micro g x mL(-1) for BPU, SBU and JBU, respectively). This platelet activating property was also independent of urease activity. Comparison of the kinetic properties indicated that SBU is fivefold less susceptible than JBU to inhibition by acetohydroxamic acid, a chelator of Ni(+2) and Zn(+2) ions. The ureases also showed different susceptibility to agents that modify cysteine residues, such as p-hydroxymercuribenzoate and p-benzoquinone. Altogether, these data emphasize that biological properties that are independent of ureolytic activity are not restricted to jackbean ureases and that these proteins may have a role in plant defense against insect predators.     

Cysteine based novel noncompetitive inhibitors of urease(s)--distinctive inhibition susceptibility of microbial and plant ureases

Based on the catalysis mechanism of urease, a homologous series of 10 cysteine derivatives (CysDs) was designed and synthesized, and their inhibitory activities were evaluated for microbial ureases (Bacillus pasteurii, BPU, and Proteus mirabilis, PMU) and for a plant urease [jack bean (Cavavalia ensiformis), JBU]. As already described, thiol-compounds might inhibit urease activity by chelating the nickel atoms involved in the catalysis process. In contrast to cysteine, which has been reported to be a very weak urease inhibitor, we verified a potential inhibitory activity of these CysDs. The kinetic data demonstrate that thiol derivatives are more effective than the respective thioether derivatives. Besides, thiol-CysDs had a reduced activity in acidic pH (5.0). Lineweaver-Burk plots indicated that the nature of inhibition was of noncompetitive type for all 10 compounds, with the minimum Ki value of 2 microM for N,N-dimethyl L-cysteine. It is proposed that these classes of compounds are more potent inhibitors of the bacterial ureases, compared with the plant-originated urease. Since microbial urease is directly involved in the infection process of many pathological organisms, this work demonstrates that thiol-CysDs represent a class of new potential urease inhibitors.     

Jack bean urease (EC 3.5.1.5) aggregation monitored by dynamic and static light scattering

Aggregation of jack bean urease (JBU) is involved in many alterations of its biological properties, notably the ureolytic and entomotoxic activities. In order to investigate this phenomenon, protein aggregates were characterized by dynamic (DLS) and static light scattering (SLS) spectroscopies through determination of apparent hydrodynamic radii, the average molecular masses, radii of gyration and second virial coefficients. No effect of disulfide reducing agents on protein association was observed contrasting with previous reports implicating their function in the prevention of JBU aggregation. The influence of freeze-thawing cycles on protein aggregation was also investigated. Our results showed that after freeze-thawing cycles the native form of JBU with apparent hydrodynamic radius of 7 nm and radius of gyration of 12 nm is replaced by high-order oligomers and this aggregation is not reverted neither by dithiothreitol (DTT) treatment nor by high concentration of salts. Altogether the data help to understand the complex behavior of JBU in solution and may correlate with the diversity of biological properties of this enzyme.     

Effects of anti-Ureaplasma urease antibody on homologous and heterologous urease activities

Among organisms in the class Mollicutes only Ureaplasma species possess urease. Antiserum to urease of U. urealyticum strain T960 (CX8) was used to examine the cross-reactivity of urease from other Ureaplasma species, as well as urease of jack bean and several urease-possessing walled bacteria. Immunological cross reactivity was used to establish phylogenetic relationships between various antigens. The ability of monospecific anti-urease antibody to inhibit urease activity was examined. The antiserum inhibited urease activity of the homologous strain the least of any Ureaplasma tested. It is postulated that urease possesses a minimum of two sets of epitopes. Binding of antibody to one epitope causes inhibition of enzyme activity; this epitope is common to urease of all Ureaplasma species. Binding of antibody to the other epitope prevents binding to the inhibition epitope; this epitope is specific to U. urealyticum strain T960 (CX8). No inhibition was observed with urease from jack bean or several walled bacteria.     

Kinetic properties of Helicobacter pylori urease compared with jack bean urease

The urease proteins of the jack bean (Canavalia ensiformis) and Helicobacter pylori are similar in molecular mass when separated by non-denaturing gradient polyacrylamide gel electrophoresis, both having three main forms. The molecular mass of their major protein form is within the range 440-480 kDa with the other two lesser forms at 230-260 kDa and 660-740 kDa. These forms are all urease active; however, significant kinetic differences exist between the H. pylori and jack bean ureases. Jack bean urease has a single pH optimum at 7.4, whereas H. pylori urease has two pH optima of 4.6 and 8.2 in barbitone and phosphate buffers that were capable of spanning the pH range 3 to 10. The H. pylori Km was 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tris.HCl at pH 8.2. By comparison, the jack bean urease had a Km of 1.3 mM in Tris.HCl under our experimental conditions. The findings show that the urease activity of H. pylori was inhibited at the pH optimum of 4.6 in the phosphate buffer, but not in the barbitone buffer. This was shown to be due to competitive inhibition by the sodium and potassium ions in the phosphate buffer, not the phosphate ions as suggested earlier. Jack bean urease activity was similarly inhibited by phosphate buffer but again due to the effect of sodium and potassium ions.     

Antiureolytic Activity of Substituted 2,5‐Diaminobenzoquinones

A series of 2,5‐bis(alkyl/arylamino)‐1,4‐benzoquinones ( 1 – 12 ) were investigated in vitro for their potential to inhibit the activity of jack bean urease. Compounds 1–6 , 8 , 9 , 11 and 12 effectively inhibited the jack bean urease activity by 90.8 % when tested at 5 μm , whereas 7 and 10 had relatively little effect. The IC₅₀ for most compounds was in the nanomolar range (31.4 nm and 36.0 nm for 2 and 8 , respectively). The mechanism of enzyme inhibition shown by 2 and 8 is typical of mixed‐type inhibitors, whose affinity for the active site is over 6‐ and 2‐fold higher (K_i=30.0 and 22.8 nm , for 2 and 8 , respectively) than that of an allosteric site. Molecular docking studies revealed that both 2 and 8 establish hydrogen bonds with the amino acids residues Asp494, Met588, His593 and Ala636 in the active site of jack bean urease. These results indicate that such aminoquinones are useful leads for the development of more efficient urease inhibitors of wider utility.     

Antifungal properties of Canavalia ensiformis urease and derived peptides

Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea into ammonia and CO₂. These proteins have insecticidal and fungicidal effects not related to their enzymatic activity. The insecticidal activity of urease is mostly dependent on the release of internal peptides after hydrolysis by insect digestive cathepsins. Jaburetox is a recombinant version of one of these peptides, expressed in Escherichia coli. The antifungal activity of ureases in filamentous fungi occurs at submicromolar doses, with damage to the cell membranes. Here we evaluated the toxic effect of Canavalia ensiformis urease (JBU) on different yeast species and carried out studies aiming to identify antifungal domain(s) of JBU. Data showed that toxicity of JBU varied according to the genus and species of yeasts, causing inhibition of proliferation, induction of morphological alterations with formation of pseudohyphae, changes in the transport of H⁺ and carbohydrate metabolism, and permeabilization of membranes, which eventually lead to cell death. Hydrolysis of JBU with papain resulted in fungitoxic peptides (∼10 kDa), which analyzed by mass spectrometry, revealed the presence of a fragment containing the N-terminal sequence of the entomotoxic peptide Jaburetox. Tests with Jaburetox on yeasts and filamentous fungi indicated a fungitoxic activity similar to ureases. Plant ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important mycoses as well as phytopathogenic fungi, especially considering their potent activity in the range of 10⁻⁶–10⁻⁷ M.     

Free vs chitosan-immobilized urease: Microenvironmental effects on enzyme inhibitions

Chitosan gel membranes were prepared by a solvent-evaporation method and used as a support for covalent immobilization of jack bean urease. The effects of the local microenvironment created by both the electrostatic potential of the polycationic support and the enzyme reaction on the inhibition of urease by phosphate buffer were investigated as a function of pH and compared with other urease competitive inhibitions. It was found that the kinetic behaviour of chitosan-immobilized urease in the inhibition is resultant of structural, diffusion limitation-related and microenvironmental factors. More importantly, it was shown that this behaviour is microenvironment-dependent when either a pH-dependence of enzyme inhibition (phosphate and F^-) or electrostatic inhibitor-support repulsion (Ni^2 +) prevailed in the system. Other inhibitors uncharged that are do not show pH-dependented inhibition (boric and acetohydroxamic acids), the inhibition only depends on factors other than microenvironmental. Knowledge of such microenvironmental effects is of practical and theoretical importance in designing applications of immobilized enzymes and in clarifying the mode of action of enzymes in biological membranes in their native milieu.     

Free vs chitosan-immobilized urease: Microenvironmental effects on enzyme inhibitions

Chitosan gel membranes were prepared by a solvent-evaporation method and used as a support for covalent immobilization of jack bean urease. The effects of the local microenvironment created by both the electrostatic potential of the polycationic support and the enzyme reaction on the inhibition of urease by phosphate buffer were investigated as a function of pH and compared with other urease competitive inhibitions. It was found that the kinetic behaviour of chitosan-immobilized urease in the inhibition is resultant of structural, diffusion limitation-related and microenvironmental factors. More importantly, it was shown that this behaviour is microenvironment-dependent when either a pH-dependence of enzyme inhibition (phosphate and F^?) or electrostatic inhibitor-support repulsion (Ni^2?+?) prevailed in the system. Other inhibitors uncharged that are do not show pH-dependented inhibition (boric and acetohydroxamic acids), the inhibition only depends on factors other than microenvironmental. Knowledge of such microenvironmental effects is of practical and theoretical importance in designing applications of immobilized enzymes and in clarifying the mode of action of enzymes in biological membranes in their native milieu.     

Multiple Intermediate Conformations of Jack Bean Urease at Low pH: Anion-induced Refolding

Structural and functional characteristics of jack bean urease (JBU), a hexameric enzyme having identical subunits, were investigated under neutral as well as acidic conditions by using CD, fluorescence, ANS binding and enzyme activity measurements. At low pH and low ionic strength, JBU exists in a partially unfolded state (U_A-state), having predominantly β structure and no tertiary interactions along with a strong ANS binding. Addition of salts like NaCl, KCl and Na₂SO₄ to the U_A-state induces refolding resulting in structural propensities similar to that of native hexamer. Moreover, at low concentrations, GuHCl behaves like an anion by inducing refolding of the U_A-state. The anion-induced refolded state (I_A-state) is more stable than U_A-state and the stability is nearly equal to that of the native protein against chemical-induced and thermal denaturation. Overall, these observations support a model of protein folding for a multimeric protein where certain conformations (ensembles of substates) of low energy prevail and populated under non-native conditions with different stability.