Concentrations Of Guanidine Hydrochloride Research Articles

Immobilization of liposomes onto quartz crystal microbalance to detect interaction between liposomes and proteins

To study the interaction between liposomes and proteins, intact liposomes were immobilized on a metal planar support by chemical binding and/or bioaffinity using a quartz crystal microbalance (QCM). A large decrease in the resonance frequency of quartz crystal was observed when the QCM, modified by a self-assembled monolayer (SAM) of carboxythiol, was added to liposome solutions. The stable chemical immobilization of intact liposomes onto SAM was judged according to the degree with which adsorbed mass depended on the prepared size of liposomes, as well as on the activation time of SAMs when amino-coupling was introduced, where the liposome coverage of electrodes was 69 ± 8 % in optimal conditions. When avidin–biotin binding was used on amino-coupling liposome layers, liposome immobilization finally reached 168% coverage of the electrode surface. Denatured protein was also successfully detected according to the change in the frequency of the liposome-immobilized QCM. The adsorbed mass of denatured carbonic anhydrase from bovine onto immobilized liposomes showed a characteristic peak at a concentration of guanidine hydrochloride that corresponded to a molten globule-like state of the protein, although the mass adsorbed onto deactivated SAM increased monotonously.

Main-chain Dominated Amyloid Structures Demonstrated by the Effect of High Pressure

It has been suggested that, while the globular native forms of proteins are a side-chain-dominated compact structure evolved by pursuing a unique fold with optimal packing of amino acid residues, amyloid fibrils are a main-chain-dominated structure with an extensive hydrogen bond network. To address this issue, the effects of hydrostatic pressure on amyloid fibrils of beta2-microglobulin (beta2-m), involved in dialysis-related amyloidosis, were studied. A systematic analysis at various pressures and concentrations of guanidine hydrochloride conducted by monitoring thioflavin T fluorescence, light-scattering, and tryptophan fluorescence revealed contrasting conformational changes occurring consecutively: first, a pressure-induced reorganization of fibrils and then a pressure-induced unfolding. The changes in volume as well as the observed structural changes indicate that the beta2-m amyloid fibrils under ambient pressure are less tightly packed with a larger number of cavities, consistent with the main-chain-dominated amyloid structure. Moreover, the amyloid structure without optimal packing will enable various isoforms to form, suggesting the structural basis of multiple forms of amyloid fibrils in contrast to the unique native-fold.

Ultrafast Events in the Folding of Ferrocytochrome c

Laser flash photolysis and stopped-flow methods have been used to study the dynamic events in the micro- to millisecond time bin in the refolding of horse ferrocytochrome c in the full range of guanidine hydrochloride concentration at pH 12.8 (+/-0.1), 22 degrees C. Under the absolute refolding condition, the earliest relaxation time of the unfolded protein chain is less than 1 micros. The chain then undergoes diffusive dynamics-mediated contraction and expansion, in which intrapolypeptide ligands make transient contacts with the heme iron, giving rise to two distinct kinetic phases of approximately 0.4 and approximately 3 micros. Under moderate to absolute refolding conditions, the rates of these processes show little dependence on the denaturant concentration, indicating the absence of structural element in the incipient or the relaxed state. Chain expansion and contraction events continue until the polypeptide finds a stable and supportive transition state. The crossing of this transition barrier, which rate-limits the folding of alkaline ferrocytochrome c, is characterized by a stopped-flow measured time constant of approximately 3 ms in aqueous solvent. Observed kinetics thus implicate no submillisecond folding structure. The folding kinetics is effectively two state in which the unfolded polypeptide first relaxes to an unstructured chain and then crosses over a late rate-limiting barrier to achieve the native conformation. The experimentally observed rates as a function of guanidine hydrochloride concentration have been simulated by numerically calculated microscopic rates of a simple kinetic model that captures the essential features of folding.

Suppression effect of guanidine hydrochloride on α-cyclodextrin-assisted refolding of denatured α-amylase

It is well established that alpha-cyclodextrin (α-CD) is an effective refolding aid for some proteins in dilution additive mode. On the other hand, it has recently been shown that guanidine hydrochloride (GuHCl), the well known protein denaturant, acts, at well defined mild concentrational ranges, as a stabilizer of the “molten globule” intermediates with the final renaturation as well as aggregation of the denatured protein under investigation. In the present study, the effect of GuHCl on α-CD efficacy on protein renaturation was investigated. The unassisted and α-CD-assisted refolding of chemically denatured α-amylase in the presence of various α-CD and GuHCl concentrations were evaluated, using activity assay, far-UV circular dichroism (CD) spectroscopy, turbidimetric analysis and hydrophobic probe 8-anilinonaphtalene-1-sulfonic acid (ANS) fluorescence techniques. The recovered activity, average helical structure and the extent of dilution-induced aggregation of refolded α-amylase were affected with increase of final GuHCl concentration in dilution buffer, especially in α-CD-assisted refolded samples. The refolding yield and protein helicity decreased, while apparent turbidity due to protein aggregation ( A lim) and its rate were increased. The results clearly indicated that α-cyclodextrin effectively suppressed aggregation, especially at lower GuHCl concentrations in the refolding buffer. Based on these results, application of low dilution factors in α-CD-dependent protein renaturation systems should be avoided due to inhibitory effects of high final denaturant concentration on α-CD efficacy besides its direct influence on the production of aggregation-prone intermediate(s) of protein molecules.

Guanidine hydrochloride denaturation of human serum albumin originates by local unfolding of some stable loops in domain III

The effect of guanidine hydrochloride (GnHCl) on the global stability of human serum albumin (HSA) has been studied by fluorescence and circular dichroism spectroscopic measurements. The differential stability of native conformation of three HSA domains were explored by using domain-specific ligands, hemin (domain I), chloroform (domain II), bilirubin (at domain I/domain II interface) and diazepam (domain III). GnHCl induced unfolding transition curves as monitored by probes for secondary and tertiary structures were cooperative but noncoincidental. A strong ANS binding to the protein was observed around 1.8 M GnHCl, suggesting existence of intermediate states in the unfolding pathway of HSA. A gradual decrease (in the GnHCl concentration range 0.0–1.8 M) in the binding of diazepam indicates that domain III is the most labile to GnHCl denaturation. A significant increase in the binding of bilirubin up to 1.4 M GnHCl and decrease thereafter leading to complete abolishment of bilirubin binding at around 2.0 M GnHCl suggest favorable rearrangement and separation of domains I and II at 1.4 and 2.0 M GnHCl concentration, respectively. Above 1.6 M GnHCl, decrease of the binding of hemin, a ligand for domain I, chloroform, which binds in domain II and lone tryptophanyl fluorescence (Trp-214 located in domain II) indicate that at higher concentration of GnHCl domains I and II start unfolding simultaneously but the stability of domain I (7.4 Kcal/mol) is much more than domain II (4.3 Kcal/mol). A pictorial model for the unfolding of HSA domains, consistent with all these results, has been formulated, suggesting that domain III is the most labile followed by domain II while domain I is the most stable. A molten globule like state of domain III around 1.8 M GnHCl has also been identified and characterized.

Measuring Unfolding of Proteins in the Presence of Denaturant Using Fluorescence Correlation Spectroscopy

IFABP is a small (15 kDa) protein consisting mostly of antiparallel β-strands that surround a large cavity into which ligands bind. We have previously used FCS to show that the native protein, labeled with fluorescein, exhibits dynamic fluctuation with a relaxation time of 35μs. Here we report the use of FCS to study the unfolding of the protein induced by guanidine hydrochloride. Although the application of this technique to measure diffusion coefficients and molecular dynamics is straightforward, the FCS results need to be corrected for both viscosity and refractive index changes as the guanidine hydrochloride concentration increases. We present here a detailed study of the effects of viscosity and refractive index of guanidine hydrochloride solutions to calibrate FCS data. After correction, the increase in the diffusion time of IFABP corresponds well with the unfolding transition monitored by far ultraviolet circular dichroism. We also show that the magnitude of the 35μs phase, reflecting the conformational fluctuation in the native state, decreases sharply as the concentration of denaturant increases and the protein unfolds. Although FCS experiments indicate that the unfolded state at pH 2 is rather compact and native-like, the radius in the presence of guanidine hydrochloride falls well within the range expected for a random coil.

Directed Evolution, Phage Display and Combination of Evolved Mutants: A Strategy to Recover the Neutralization Properties of the scFv Version of BCF2 a Neutralizing Monoclonal Antibody Specific to Scorpion Toxin Cn2

BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.

Apparent Debye−Huckel Electrostatic Effects in the Folding of a Simple, Single Domain Protein

We have monitored the effects of salts and denaturants on the folding of the simple, two-state protein FynSH3. As predicted by Debye-Huckel limiting law, both the stability and (log) folding rate of FynSH3 increase nearly perfectly linearly (r(2)> 0.99) with the square root of ionic strength upon increasing concentrations of the relatively nonchaotropic salt sodium chloride. The stability of FynSH3 is also linear in square root ionic strength when the relatively nonchaotropic salts sodium bromide, potassium bromide, and potassium chloride are employed. Comparison of the kinetic and equilibrium effects of sodium chloride suggests that the electrostatic interactions formed in the folding transition state are approximately 50% as destabilizing as those formed in the native state, presumably reflecting the more compact nature of the latter. In contrast, the relationship between concentration and folding kinetics is more complex when the highly chaotropic salt guanidine hydrochloride (GuHCl) is employed. At moderate to high GuHCl concentrations the net effect of the linear, presumably chaotrope-induced deceleration and the presumed, square root-dependent ionic strength-induced acceleration is well approximated as linear, thus accounting for the observation of "chevron behavior" (log folding rate linear in denaturant concentration) typically reported for the folding of single domain proteins. At very low GuHCl concentrations, however, significant kinetic rollover is observed. This rollover is reasonably well fitted as a sum of a linear, presumably chaotropic effect and a square root-dependent, presumably electrostatic effect. These results thus not only provide insight into the nature of the folding transition state but also suggest that caution is in order when extrapolating GuHCl-based chevrons to estimate folding rates in the absence of denaturant and in interpreting deviations from chevron linearity as evidence for non-two-state kinetics.

The effect of concentration of guanidine hydrochloride on the sulfasalazine–serum albumin complex

A detailed study of the effect of Gu·HCl concentration is presented in this work. Destabilization of the tertiary structure of BSA was observed by using fluorescence measurements of excitation wavelength of 280 and 295 nm. We ascertained that in terms of the effect on the fluorescence emission spectrum of BSA, Gu·HCl concentration falls into three regions: I, 0.1–1.6 M; II, 1.6–3 M and III, 3–6 M. The intermediate state of BSA denaturation for 1.6–1.7 M Gu·HCl was noticed. Quenching of the native BSA fluorescence by sulfasalazine (SSZ), a drug used in inflammatory bowel disease, was shown. In the presence of Gu·HCl, BSA fluorescence rises or decreases depending on the concentration region I or II, respectively. A similar effect of Gu·HCl on the fluorescence spectrum of BSA, quenched by sulfasalazine, can be found. However, Gu·HCl concentration-dependent reduction of the quenching effect of the SSZ points to the gradual loss of the binding properties. Binding and quenching constants for the SSZ–BSA complexes were calculated in the absence and presence of the denaturant. Since Gu·HCl at concentration 0.1–1.6 M increases and at concentration 1.6–3 M decreases the BSA fluorescence, the mechanism of the destabilization of the native structure of serum albumin differs for these two ranges of Gu·HCl concentration. The small concentration of the denaturant causes a disorder in the hydration layer of the albumin and the higher one causes the binding of the denaturant molecule close to the Trp 135 in the IB subdomain.

FMN binding and unfolding of Desulfovibrio desulfuricans flavodoxin: “hidden” intermediates at low denaturant concentrations

The flavin mononucleotide (FMN) cofactor in Desulfovibrio desulfuricans flavodoxin stays associated with the polypeptide upon guanidine hydrochloride (GuHCl) induced unfolding. Using isothermal titration calorimetry (ITC), we determined the affinity of FMN for the flavodoxin polypeptide as a function of both urea and GuHCl concentrations (pH 7, 25 °C). The FMN affinity for folded and GuHCl-unfolded flavodoxin differs 10-fold, which is in agreement with the difference in thermodynamic stability between the apo- and holo-forms. In contrast, the urea-unfolded protein does not interact with FMN and equilibrium unfolding of holo-flavodoxin in urea results in FMN dissociation prior to polypeptide unfolding. ANS-binding, near-UV circular dichroism (CD), acrylamide quenching and FMN-emission experiments reveal the presence of native-like intermediates, not detected by far-UV CD and aromatic fluorescence detection methods, in low concentrations of both denaturants. Time-resolved experiments show that FMN binding is fastest at GuHCl concentrations where the native-like intermediate species is populated.

The studies of FT-IR and CD spectroscopy on catechol oxidase I from tobacco

A novel copper-containing enzyme named COI (catechol oxidase I) has been isolated and purified from tobacco by extracting acetone-emerged powder with phosphate buffer, centrifugation at low temperature, ammonium sulfate fractional precipitation, and column chromatography on DEAE-sephadex (A-50), sephadex (G-75), and DEAE-celluse (DE-52). PAGE, SDS-PAGE were used to detect the enzyme purity, and to determine its molecular weight. Then the secondary structures of COI at different pH, different temperatures and different concentrations of guanidine hydrochloride (GdnHCl) were studied by the FT-IR, Fourier self-deconvolution spectra, and circular dichroism (CD). At pH 2.0, the contents of both α-helix and anti-parallel β-sheet decrease, and that of random coil increases, while β-turn is unchanged compared with the neutral condition (pH 7.0). At pH 11.0, the results indicate that the contents of α-helix, anti-parallel β-sheet and β-turn decrease, while random coil structure increases. According to the CD measurements, the relative average fractions of α-helix, anti-parallel β-sheet, β-turn/parallel β-sheet, aromatic residues and disulfide bond, and random coil/γ-turn are 41.7%, 16.7%, 23.5%, 11.3%, and 6.8% at pH 7.0, respectively, while 7.2%, 7.7%, 15.2%, 10.7%, 59.2% at pH 2.0, and 20.6%, 9.5%, 15.2%, 10.5%, 44.2% at pH 11.0. Both α-helix and random coil decrease with temperature increasing, and anti-parallel β-sheet increases at the same time. After incubated in 6 mol/L guanidine hydrochloride for 30 min, the fraction of α-helix almost disappears (only 1.1% left), while random coil/γ-turn increases to 81.8%, which coincides well with the results obtained through enzymatic activity experiment.

Effects of Heme on the Structure of the Denatured State and Folding Kinetics of Cytochrome b562

Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-alpha-helical bundle. The holo protein apparently refolds with a half-life of 4 micros in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 micros that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins.

Cloning, expression, and refolding of a secretory protein ESAT-6 of Mycobacterium tuberculosis

A DNA encoding the 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis was inserted into a bacterial expression vector of pQE30 resulting in a 6× His- esat-6 fusion gene construction. This plasmid was transformed into Escherichia coli strain M15 and effectively expressed. The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8 M urea or 6 M guanidine–hydrochloride at pH 7.4, and the recombinant protein was purified by Ni–NTA column. The purified fusion protein was refolded by dialysis with a gradient of decreasing concentration of urea or guanidine hydrochloride or by the size exclusion protein refolding system. The yield of refolded protein obtained from urea dialysis was 20 times higher than that from guanidine–hydrochloride. Sixty-six percent of recombinant ESAT-6 was successfully refolded as monomer protein by urea gradient dialysis, while 69% of recombinant ESAT-6 was successfully refolded as monomer protein by using Sephadex G-200 size exclusion column. These results indicate that urea is more suitable than guanidine–hydrochloride in extracting and refolding the protein. Between the urea gradient dialysis and the size exclusion protein refolding system, the yield of the monomer protein was almost the same, but the size exclusion protein refolding system needs less time and reagents.

Protein Stiffening and Entropic Stabilization in the Subdenaturing Limit of Guanidine Hydrochloride

Subdenaturing concentrations of guanidine hydrochloride (GdnHCl) stabilize proteins. For ferrocytochrome c the stabilization is detected at subglobal level with no measured change in global stability. These deductions are made by comparing observed rates of thermally driven ferrocytochrome cHCO reactions with global unfolding rates of ferrocytochrome c measured by stopped flow and NMR hydrogen exchange in the presence of a wide range of GdnHCl concentrations at pH 7, 22 °C.

Ligand Binding Subsequent to NO Photolyses of Partially Unfolded Cytochrome c

AbstractHere we report folding studies of horse heart ferricytochrome c in the presence of NO (NO‐cyt c). The midpoint concentration of guanidine hydrochloride (GuHCl) for unfolding was measured to be 2.8 M in the presence of NO and is only moderately altered relative to native cyt c under similar conditions (i.e., 2.6 M GuHCl). Recombination of NO to cyt c in the presence of 2.8 M GuHCl subsequent to photolysis is biphasic with rate constants of (4.24 ± 0.15) × 103 s−1 and 197.7 ± 3.3 s−1 with the fast and slow phase representing ˜60% and 40% relative amplitudes, respectively. NO rebinding to unfolded cyt c (in the presence of 4 M GuHCl) exhibits two phases with rate constants similar to those observed in the presence of 2.8 M GuHCl with the relative amplitude of the fast phase increasing to roughly 90%. These data suggest that NO rebinds to two populations of cyt c with one population containing a five coordinate heme and the other having a six‐coordinate heme with bis‐His axial ligation. These results suggest that the mis‐ligated bis‐His conformation forms rapidly during the refolding of cyt. c3+, although a population exists that can refold down a native pathway.

Extracellular nuclease from a thermophile, Streptomyces thermonitrificans: production, purification and partial characterization of – double strand preferential – deoxyribonuclease activity

A thermophilic strain of Streptomyces thermonitrificans produced high levels of extracellular nuclease (designated as nuclease Stn β) when grown on nutrient broth glucose medium. Maximal nuclease activity (51 U ml −1) was obtained, in 40 h, when the culture was grown on modified NBG medium at 45 °C. The enzyme was purified to homogeneity with an overall recovery of 5.6% and a specific activity of 10,833. The relative molecular mass of the purified enzyme, determined by gel filtration, was 22.4 kDa and it showed an obligate requirement for Mn 2+ for activity. The optimum pH and temperature of nuclease Stn β were 8.0 and 45 °C, respectively. The enzyme was inhibited by Mg 2+, Co 2+, Cu 2+, Zn 2+ and Hg 2+, inorganic phosphate, pyrophosphate, dithiothreitol, β-mercaptoethanol, EDTA and NaCl. Nuclease Stn β was stable to high concentrations of urea and organic solvents but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Stn β is a multifunctional enzyme with substrate specificity in the order of dsDNA > ssDNA ≫ RNA. The end products of dsDNA hydrolysis were predominantly oligonucleotides (85–90%) and small amounts of 5′ mononucleotides (10–15%) suggesting an endo mode of action.

Estimating the energetic contribution of hydrogen bonding to the stability of Candida methylica formate dehydrogenase by using double mutant cycle

An homology model of Candida methylica formate dehydrogenase (cm FDH) was constructed based on the Pseudomonas sp. 101 formate dehydrogenase (ps FDH) structure. In wild type cm FDH, Thr169 and Thr226 can form hydrogen bonds with each other. We measured the interaction energy between the two threonines independent of other interactions in the proteins by using a so-called double mutant cycle and assessing the protein stability from the concentration of guanidine hydrochloride needed to denature 50% of the molecules. We conclude that the hydrogen bonds stabilize the wild type protein by -4 kcal mol(-1).

Guanidine hydrochloride can induce amyloid fibril formation from hen egg-white lysozyme.

The formation of amyloid fibrils is an intractable problem in which normally soluble protein polymerizes and forms insoluble ordered aggregates. Such aggregates can range from being a nuisance in vitro to being toxic in vivo. The latter is true for lysozyme, which has been shown to form toxic deposits in humans. In the present study, the effects of partial denaturation of hen egg-white lysozyme via incubation in a concentrated solution of the denaturant guanidine hydrochloride are investigated. Results show that when lysozyme is incubated under moderate guanidine hydrochloride concentrations (i.e., 2-5 M), where lysozyme is partially unfolded, fibrils form rapidly. Thioflavin T, Congo red, X-ray diffraction, transmission electron microscopy, atomic force microscopy, and circular dichroism spectroscopy are all used to verify the production of fibrils under these conditions. Incubation at very low or very high guanidine hydrochloride concentrations fails to produce fibrils. At very low denaturant concentrations, the structure of lysozyme is fully native and very stable. On the other hand, at very high denaturant concentrations, guanidine hydrochloride is capable of dissolving and dis-aggregating fibrils that are formed. Raising the temperature and/or concentration of lysozyme accelerates fibril formation by further adding to the concentration of partially unfolded species. The addition of preformed fibrils also accelerates fibril formation but only under partially unfolding conditions. The results presented here provide further evidence that partial unfolding is a prerequisite to fibril formation. Partial denaturation can accelerate fibril formation in much the same way that mutations have been shown to accelerate fibril formation.

The conformational stability of the Streptomyces coelicolor histidine-phosphocarrier protein. Characterization of cold denaturation and urea-protein interactions.

Thermodynamic parameters describing the conformational stability of the histidine-containing phosphocarrier protein from Streptomyces coelicolor, scHPr, have been determined by steady-state fluorescence measurements of isothermal urea-denaturations, differential scanning calorimetry at different guanidinium hydrochloride concentrations and, independently, by far-UV circular dichroism measurements of isothermal urea-denaturations, and thermal denaturations at fixed urea concentrations. The equilibrium unfolding transitions are described adequately by the two-state model and they validate the linear free-energy extrapolation model, over the large temperature range explored, and the urea concentrations used. At moderate urea concentrations (from 2 to 3 m), scHPr undergoes both high- and low-temperature unfolding. The free-energy stability curves have been obtained for the whole temperature range and values of the thermodynamic parameters governing the heat- and cold-denaturation processes have been obtained. Cold-denaturation of the protein is the result of the combination of an unusually high heat capacity change (1.4 +/- 0.3 kcal.mol(-1).K(-1), at 0 m urea, being the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements) and a fairly low enthalpy change upon unfolding at the midpoint temperature of heat-denaturation (59 +/- 4 kcal.mol(-1), the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements). The changes in enthalpy (m(DeltaH(i) )), entropy (m(DeltaS(i) )) and heat capacity (m(DeltaC(pi) )), which occur upon preferential urea binding to the unfolded state vs. the folded state of the protein, have also been determined. The m(DeltaH(i) ) and the m(DeltaS(i) ) are negative at low temperatures, but as the temperature is increased, m(DeltaH(i) ) makes a less favourable contribution than m(DeltaS(i) ) to the change in free energy upon urea binding. The m(DeltaC(pi) ) is larger than those observed for other proteins; however, its contribution to the global heat capacity change upon unfolding is small.

Low concentration of guanidine hydrochloride induces the formation of an aggregation-prone state in alpha-urease.

Canavalia ensiformis (jack bean) alpha-urease is a hexameric protein characterized by a complex denaturation mechanism. In previous papers, we have shown that a hydrophobic 8-anilino-1-naphthalenesulfonic acid (ANSA) binding conformer could be populated in a moderate concentration of denaturant. This state was obtained under conditions that had no detectable impact on its tertiary structure, as indicated by fluorescence measurements. In the present study, we further characterized this ANSA-binding state in an attempt to understand urease behavior. Evidence presented here shows that the presence of ANSA was not required for the generation of the conformer and that its affinity for ANSA came from an increase in hydrophobicity leading to aggregation. Circular dichroism investigation of urease revealed that it had periodical secondary structure content similar to Klebsiella aerogenes urease (secondary structures calculated on the basis of crystallographic data). The impact of 0.9 M guanidine hydrochloride (GuHCl) on soluble urease secondary structures was minimal but is compatible with a slight increase in beta-sheet structures. Such modification may indicates that aggregation involves amyloid-like fibril formation. Electron microscopy analysis of urease in the absence of GuHCl revealed the presence of urease hexamers (round shape 13 nm in diameter). These particles disappeared in the presence of moderate denaturant concentration owing to the formation of aggregates and fibril-like structures. The fibrils obtained in 1.5 M GuHCl had an average diameter of 6.5 nm, suggesting that urease hexamers dissociated into smaller oligomeric forms when forming such fibrils.