Reshaping the folding energy landscape by chloride salt: impact on molten-globule formation and aggregation behavior of carbonic anhydrase

During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used. Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl. On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea). However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl. Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa). Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt. Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state. Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase. Refolding from this state only provided low recoveries of native enzyme.     

Cofactor effects on the protein folding reaction: acceleration of alpha-lactalbumin refolding by metal ions

About 30% of proteins require cofactors for their proper folding. The effects of cofactors on the folding reaction have been investigated with alpha-lactalbumin as a model protein and metal ions as cofactors. Metal ions accelerate the refolding of alpha-lactalbumin by lessening the energy barrier between the molten globule state and the transition state, mainly by decreasing the difference of entropy between the two states. These effects are linked to metal ion binding to the protein in the native state. Hence, relationships between the metal affinities for the intermediate states and those for the native state are observed. Some residual specificity for the calcium ion is still observed in the molten globule state, this specificity getting closer in the transition state to that of the native state. The comparison between kinetic and steady-state data in association with the Phi value method indicates the binding of the metal ions on the unfolded state of alpha-lactalbumin. Altogether, these results provide insight into cofactor effects on protein folding. They also suggest new possibilities to investigate the presence of residual native structures in the unfolded state of protein and the effects of such structures on the protein folding reaction and on protein stability.     

Metal ion-induced stabilization and refolding of anticoagulation factor II from the venom of Agkistrodon acutus

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF II, holo-ACF II, and Tb(3+)-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism (CD). Metal ions were found to increase the structural stability of ACF II against GdnHCl and irreversible thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF II and Tb(3+)-ACF II is a two-state process with no detectable intermediate state, while the GdnHCl-induced unfolding/refolding of holo-ACF II in the presence of 1 mM Ca(2+) follows a three-state transition with an intermediate state. Ca(2+) ions play an important role in the stabilization of both native and I states of holo-ACF II. The decalcification of holo-ACF II shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, while the reconstitution of apo-ACF II with Tb(3+) ions shifts the initial zone of the denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.1 M GdnHCl) at which refolding from the fully denatured state of apo-ACF II to the I state of holo-ACF II or to the native state of Tb(3+)-ACF II can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF II, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ion-induced refolding provide evidence for the fact that the first phase of Tb(3+)-induced refolding should involve the formation of the compact metal-binding site regions, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

Unfolding and refolding of human glyoxalase II and its single-tryptophan mutants.

Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding. Human glyoxalase II contains two tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule. Trp57 is a non-conserved residue located within a "zinc binding motif" (T/SHXHX57DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent beta-lactamase and arylsulfatase. Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates. The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic fluorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II. At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained. The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions. The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding. The comparison of unfolding fluorescence transitions of single-trypthophan mutants with that of wild-type enzyme indicates that the strictly conserved "zinc binding motif" is located in a flexible region of the active site in which Zn(II) participates in catalysis.     

Intermediate studies on refolding of arginine kinase denatured by guanidine hydrochloride.

The refolding course and intermediate of guanidine hydrochloride (GuHCl)-denatured arginine kinase (AK) were studied in terms of enzymatic activity, intrinsic fluorescence, 1-anilino-8-naphthalenesulfonte (ANS) fluorescence, and far-UV circular dichroism (CD). During AK refolding, the fluorescence intensity increased with a significantly blue shift of the emission maximum. The molar ellipticity of CD increased to close to that of native AK, as compared with the fully unfolded AK. In the AK refolding process, 2 refolding intermediates were observed at the concentration ranges of 0.8-1.0 mol/L and 0.3-0.5 mol GuHCl/L. The peak position of the fluorescence emission and the secondary structure of these conformation states remained roughly unchanged. The tryptophan fluorescence intensity increased a little. However, the ANS fluorescence intensity significantly increased, as compared with both the native and the fully unfolded states. The first refolding intermediate at the range of 0.8-1.0 mol GuHCl/L concentration represented a typical "pre-molten globule state structure" with inactivity. The second one, at the range of 0.3-0.5 mol GuHCl/L concentration, shared many structural characteristics of native AK, including its secondary and tertiary structure, and regained its catalytic function, although its activity was lower than that of native AK. The present results suggest that during the refolding of GuHCl-denatured AK there are at least 2 refolding intermediates; as well, the results provide direct evidence for the hierarchical mechanism of protein folding.     

Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies

The refolding rate of heat-denatured cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus has been reported to be unusually slow under some conditions. To elucidate the structural basis of the unusually slow kinetics of the protein, the denaturation and refolding processes of the PCP-0SH were investigated using a real-time 2D (1)H-(15)N HSQC and CD experiments. At 2 M urea denaturation of the PCP-0SH in the acidic region, all of the native peaks in the 2D HSQC spectrum completely disappeared. The conformation of the PCP-0SH just after removal of 6 M GuHCl could be observed as a stable intermediate (D(1) state) in 2D HSQC and CD experiments, which is similar to a molten globule structure. The D(1) state of the PCP-0SH, which is the initial state of refolding, corresponded to the state at 2 M urea and seemed to be the denatured state in equilibrium with the native state under the physiological conditions. The refolding of PCP-0SH from the D(1) state to the native state could be observed to be highly cooperative without any intermediates between them, even if the refolding rate was quite slow. In the higher concentration of denaturants, PCP-0SH showed HSQC and CD spectra characteristic of completely unfolded proteins called the D(2) state. The unusually slow refolding rate was discussed as originating in the conformations in the transition state and/or the retardation of reorganization in an ensemble of nonrandom denatured structures in the D(1) state.     

The role of the metal ion in the refolding of denatured bovine Co(II)-carbonic anhydrase II

Conditions for reactivation of guanidine-HCl-denatured bovine Co(II)-carbonic anhydrase II are given. The renaturation is accompanied by recovery of the native Co(II)-spectrum of the enzyme. After studying the kinetics of the renaturation process, the metal ion involvement in the refolding pathway can be summarized as follows: (1) Formation of an inactive Co(II)-intermediate with the metal ion firmly bound. No native Co(II)-spectrum is observed in this state, probably due to octahedral coordination of the metal ion in this intermediate. (2) Formation of an inactive Co(II)-intermediate with a native Co(II)-spectrum. The final tetrahedral coordination of the metal ion seems to have been formed in this state. (3) Formation of the active conformation of the enzyme. A functioning active-site is formed after some rearrangements of the polypeptide chain. This isomerisation step does not need to be preceded by formation of the intermediate with a native Co(II)-spectrum. Coordination of Co2+ in a native-like manner is, however, a prerequisite for enzymic activity. It is tentatively suggested that the metal ion is involved in stabilizing a nucleation structure formed at the bottom of the active centre. This probably occurs through binding of Co2+ to some or all of its histidyl ligands in this region after an early structuration of the metal ion binding site. The mechanisms of Co2+ appear to be similar for the refolding enzyme and the native apoenzyme, inferring that the binding site formed as a result of the nucleation process probably has the same structure as in the native conformation.     

Effect of hydrostatic pressure on unfolding of alpha-lactalbumin: volumetric equivalence of the molten globule and unfolded state

The effect of pressure on the unfolding of bovine alpha-lactalbumin was investigated by ultraviolet absorption methods. The change of molar volume associated with unfolding, deltaV, was measured in the presence or absence of guanidine hydrochloride at pH 7. The deltaV was estimated to be -63 cm3/mol in the absence of a chemical denaturant. While in the presence of guanidine hydrochloride (GuHCl), it was found that deltaV was -66 cm3/mol at 25 degrees C and was independent of the concentration of GuHCl, despite the fact that the molten globule fraction in the total unfolding product decreased with the increase of GuHCl concentration. The results indicate that the volume of alpha-lactalbumin only changes at the transition from a native to a molten globule state, and almost no volume change has been found during the transition from a molten globule to the unfolded state.     

Ca(II)- and Tb(III)-induced stabilization and refolding of anticoagulation factor I from the venom of Agkistrodon acutus

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF I, holo-ACF I, and Tb(3+)-reconstituted ACF I in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism. Metal ions were found to increase the structural stability of ACF I against GdnHCl and thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF I and Tb(3+)-ACF I is a two-state process with no detectable intermediate state(s), whereas the GdnHCl-induced unfolding/refolding of holo-ACF I in the presence of 1 mM Ca(2+) follows a three-step transition, with intermediate state a (Ia) and intermediate state b (Ib). Ca(2+) ions play an important role in the stabilization of the Ia and Ib states. The decalcification of holo-ACF I shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, whereas the reconstitution of apo-ACF I with Tb(3+) ions shifts the initial zone of denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.0 M GdnHCl) at which refolding from the fully denatured state of apo-ACF I to the Ib state of holo-ACF I or to the native state of Tb(3+)-ACF I can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF I, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ions-induced refolding provide evidence that the compact Tb(3+)-binding region forms first, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

Transient dimer in the refolding kinetics of cytochrome c characterized by small-angle X-ray scattering

The equilibrium unfolding and the kinetic refolding of cytochrome c (Cyt c) in the presence of imidazole were studied with small-angle X-ray scattering (SAXS). The equilibrium unfolding experiments showed the radius of gyration, R(g), of native Cyt c to swell approximately 1 A with the addition of imidazole. The thermodynamic parameter m also reflects an expansion of the protein as its lower value demonstrates an increase in solvent-accessible surface area over that of native Cyt c in the absence of imidazole. Refolding was studied in the presence of imidazole as it prevents misligated intermediate states from forming during the refolding process, simplifying the kinetics, and making them easier to resolve. Time-resolved decreases in the forward scattering amplitude, I(0), demonstrated the transient formation of an aggregated intermediate. Final protein and denaturant concentrations were varied in the refolding kinetics, and the singular value decomposition (SVD) method was employed to characterize the associated state. This state was determined to be a dimer, with properties consistent with a molten globule.     

Two slow stages in refolding of bovine carbonic anhydrase B are due to proline isomerization

Kinetics of refolding of bovine carbonic anhydrase B have been studied by the "double-jump" technique (i.e. the dependence of protein refolding on delay time in the unfolded state after fast unfolding). It is shown that two stages (the slow with a relaxation time of t1/2 approximately equal to 120 s and the superslow with t1/2 approximately equal to 600 s) observed during refolding of bovine carbonic anhydrase B are due to trans-cis isomerization of proline residues. The dependences of rate constants of these processes on temperature and on the final denaturant concentration were measured. Activation energies of both processes are the same, Ea = 18(+/- 2) kcal/mol. The rate constants of protein refolding do not depend on the final concentration of urea under native conditions. In addition, the rate of isomerization of essential proline residues in the "molten globule" intermediate state of bovine carbonic anhydrase was measured and found to be equal to that for unstructural polypeptides. The effect of several proline residues on carbonic anhydrase refolding is discussed.     

Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies.

Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.     

Transient association of the first intermediate during the refolding of bovine carbonic anhydrase B.

Many proteins which aggregate during refolding may form transiently populated aggregated states which do not reduce the final recovery of active species. However, the transient association of a folding intermediate will result in reduced refolding rates if the dissociation process occurs slowly. Previous studies on the refolding and aggregation of bovine carbonic anhydrase B (CAB) have shown that the molten globule first intermediate on the CAB folding pathway will form dimers and trimers prior to the formation of large aggregates (Cleland, J. L.; Wang, D. I. C. Biochemistry 1990, 29, 11072-11078; Cleland, J. L.; Wang, D. I. C. In Protein Refolding; Georgiou, G., De-Bernardez-Clark, E., Eds.; ACS Symposium Series 470; American Chemical Society: Washington, DC, 1991; pp 169-179). Refolding of CAB from 5 M guanidine hydrochloride (GuHCl) was achieved at conditions ([CAB]f = 10-33 microM, [GuHCl]f = 1.0 M) which allowed complete recovery of active protein as well as the formation of a transiently populated dimer of the molten globule intermediate on the refolding pathway. A kinetic analysis of CAB refolding provided insight into the mechanism of the association phenomenon. Using the kinetic results, a model of the refolding with transient association was constructed. By adjusting a single variable, the dimer dissociation rate constant, the model prediction fit both the experimentally determined active protein and dimer concentrations. The model developed in this analysis should also be applicable to the refolding of proteins which have been observed to form aggregates during refolding. In particular, the transient association of hydrophobic folding intermediates may also occur during the refolding of other proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of on- and off-pathway intermediates in the folding kinetics of Azotobacter vinelandii apoflavodoxin

The folding kinetics of the 179-residue Azotobacter vinelandii apoflavodoxin, which has an alpha-beta parallel topology, have been followed by stopped-flow experiments monitored by fluorescence intensity and anisotropy. Single-jump and interrupted refolding experiments show that the refolding kinetics involve four processes yielding native molecules. Interrupted unfolding experiments show that the two slowest folding processes are due to Xaa-Pro peptide bond isomerization in unfolded apoflavodoxin. The denaturant dependence of the folding kinetics is complex. Under strongly unfolding conditions (>2.5 M GuHCl), single exponential kinetics are observed. The slope of the chevron plot changes between 3 and 5 M denaturant, and no additional unfolding process is observed. This reveals the presence of two consecutive transition states on a linear pathway that surround a high-energy on-pathway intermediate. Under refolding conditions, two processes are observed for the folding of apoflavodoxin molecules with native Xaa-Pro peptide bond conformations, which implies the population of an intermediate. The slowest of these two processes becomes faster with increasing denaturant concentration, meaning that an unfolding step is rate-limiting for folding of the majority of apoflavodoxin molecules. It is shown that the intermediate that populates during refolding is off-pathway. The experimental data obtained on apoflavodoxin folding are consistent with the linear folding mechanism I(off) <==> U <==> I(on) <== > N, the off-pathway intermediate being the molten globule one that also populates during equilibrium denaturation of apoflavodoxin. The presence of such on-pathway and off-pathway intermediates in the folding kinetics of alpha-beta parallel proteins is apparently governed by protein topology.     

Effects of rare earth ions on the conformational stability of anticoagulation factor II from Agkistrodon acutus venom probed by fluorescent spectroscopy

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding of rare earth ions (RE(3+))-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solution was studied by fluorescence. The GdnHCl-induced unfolding of RE(3+) (Nd(3+), Sm(3+), Eu(3+), Gd(3+))-reconstituted ACF II follows a three-state transition with a stable intermediate state. Substitutions of the RE(3+) ions for Ca(2+) in ACF II decrease the conformational stability of its native state but markedly increase the conformational stability of its intermediate state. The free energy change of RE(3+)-ACF II from the intermediate state to denatured state linearly increases with the increase of ionic potentials of bound metal ions (Ca(2+), Nd(3+), Sm(3+), Eu(3+), and Gd(3+)). The refolding of ACF II from the unfolded state to the intermediate state can be induced merely by adding 10 microM RE(3+) ions without changing the concentration of the denaturant. The kinetic results of the RE(3+)-induced refolding provide evidence indicating that the intermediate state of RE(3+)-ACF II consists of at least two refolding phases and that the refolding rate constant values of the faster phase decrease with the increase of the difference between the radii of Ca(2+) and RE(3+), but the refolding rate constant values of the slower phase are similar to each other. The results of this study indicate that the size of metal ion is the major factor responsible for the metal ion-induced conformational stabilization of the native ACF II, while the metal ionic potential plays a predominant role in stabilizing the conformation for the intermediate state.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Initial protein concentration and residual denaturant concentration strongly affect the batch refolding of hen egg white lysozyme

The effects of several variables on the refolding of hen egg white lysozyme have been studied. Lysozyme was denatured in both urea, and guanidine hydrochloride (GuHCl), and batch refolded by dilution (100 to 1000 fold) into 0.1M Tris-HCl, pH 8.2, 1 mM EDTA, 3 mM reduced glutathione and 0.3 mM oxidised glutathione. Refolding was found to be sensitive to temperature, with the highest refolding yield obtained at 50°C. The apparent activation energy for lysozyme refolding was found to be 56 kJ/mol. Refolding by dilution results in low concentrations of both denaturant and reducing agent species. It was found that the residual concentrations obtained during dilution (100-fold dilution: [GuHCl]=0.06 mM, [DTT]=0.15 mM) were significant and could inhibit lysozyme refolding. This study has also shown that the initial protein concentration (1–10 mg/mL) that is refolded is an important parameter. In the presence of residual GuHCl and DTT, higher refolding yields were obtained when starting from higher initial lysozyme concentrations. This trend was reversed when residual denaturant components were removed from the refolding buffer.     

Intrinsically disordered structure of Bacillus pasteurii UreG as revealed by steady-state and time-resolved fluorescence spectroscopy

UreG is an essential protein for the in vivo activation of urease. In a previous study, UreG from Bacillus pasteurii was shown to behave as an intrinsically unstructured dimeric protein. Here, intrinsic and extrinsic fluorescence experiments were performed, in the absence and presence of denaturant, to provide information about the form (fully folded, molten globule, premolten globule, or random coil) that the native state of BpUreG assumes in solution. The features of the emission band of the unique tryptophan residue (W192) located on the C-terminal helix, as well as the rate of bimolecular quenching by potassium iodide, indicated that, in the native state, W192 is protected from the aqueous polar solvent, while upon addition of denaturant, a conformational change occurs that causes solvent exposure of the indole side chain. This structural change, mainly affecting the C-terminal helix, is associated with the release of static quenching, as shown by resolution of the decay-associated spectra. The exposure of protein hydrophobic sites, monitored using the fluorescent probe bis-ANS, indicated that the native dimeric state of BpUreG is disordered even though it maintains a significant amount of tertiary structure. ANS fluorescence also indicated that, upon addition of a small amount of GuHCl, a transition to a molten globule state occurs, followed by formation of a pre-molten globule state at a higher denaturant concentration. The latter form is resistant to full unfolding, as also revealed by far-UV circular dichroism spectroscopy. The hydrodynamic parameters obtained by time-resolved fluorescence anisotropy at maximal denaturant concentrations (3 M GuHCl) confirmed the existence of a disordered but stable dimeric protein core. The nature of the forces holding together the two monomers of BpUreG was investigated. Determination of free thiols in native or denaturant conditions, as well as light scattering experiments in the absence and presence of dithiothreitol as a reducing agent, under native or denaturing conditions, indicates that a disulfide bond, involving the unique conserved cysteine C68, is present under native conditions and maintained upon addition of denaturant. This covalent bond is therefore important for the stabilization of the dimer under native conditions. The intrinsically disordered structure of UreG is discussed with respect to the role of this protein as a chaperone in the urease assembly system.     

Guanidinium chloride induced unfolding of a hemocyanin subunit from Carcinus aestuarii. I. Apo form

The effects of guanidinium chloride (GuHCl) on the stability of the apo form of the 5S non-reassociating subunit of hemocyanin from the crab Carcinus aestuarii (apo-CaeSS2) were investigated, using a variety of optical spectroscopy techniques (light scattering (LS), fluorescence (IF and EF) and circular dichroism (CD)). The fluorescence of 8-anilino-1-naphtalene sulphonate (ANS) was strongly enhanced in the presence of apo-CaeSS2, in contrast to holo-CaeSS2, suggesting the formation of a molten globule (MG)-like state, consequent to the removal of the two copper ions from the holo subunit. Other evidences, favouring the presence of this state in apo-CaeSS2, derive from an enhanced quenching of intrinsic fluorescence (IF) by acrylamide, a higher sensibility towards aggregation and a higher IF with respect to deoxy holo-CaeSS2. Aggregation of apo-CaeSS2 below 1.2 M GuHCl was detected by LS, suggesting the formation of an aggregation-prone intermediate, called I1. Due to this effect, fluorescence and CD data could only be collected above that denaturant concentration. Both IF (protein) and EF (ANS) fluorescence data were best fitted by a two-state cooperative transition, occurring between the intermediate I1 and the unfolded state U, with C(1/2) 1.6-1.7 M. A similar two-state transition, with a slightly higher C(1/2) value (1.9 M), was also inferred from far-UV CD data, suggesting the possible formation of another intermediate. Partial refolding of apo-CaeSS2 by dilution was found to occur above 1.2 M GuHCl, i.e. up to the level of I1, since at lower denaturant concentration protein aggregation took place, as also observed in unfolding. All thermodynamic parameters, derived from data above 1.2 M GuHCl, are therefore referred to transitions between intermediate and unfolded states only. Unfolding kinetics, followed by fluorescence stopped-flow, was biphasic in the whole GuHCl range investigated (3-5 M), suggesting the formation of a transient intermediate, possibly related to that observed under equilibrium conditions.     

Folding-unfolding equilibrium and kinetics of equine beta-lactoglobulin: equivalence between the equilibrium molten globule state and a burst-phase folding intermediate

The denaturant-induced equilibrium unfolding transition of equine beta-lactoglobulin was investigated by ultraviolet absorption, fluorescence, and circular dichroism (CD) spectra. An equilibrium intermediate populates at moderate denaturant concentrations, and its CD spectrum is similar to that of the molten globule state previously observed for this protein at acid pH [Ikeguchi, M., Kato, S., Shimizu, A., and Sugai, S. (1997) Proteins: Struct., Funct., Genet. 27, 567-575]. The unfolding and refolding kinetics were also investigated by the stopped-flow CD and fluorescence. A significant change in the CD intensity was observed within the dead time of measurements (25 ms) when the refolding reaction was initiated by diluting the urea-unfolded protein solution, indicating the transient accumulation of the folding intermediate. The CD spectrum of this burst-phase intermediate agrees well with that of the molten globule state at acid pH. The stability of the burst-phase intermediate was also estimated from the urea-concentration dependence of the burst-phase amplitude, and it shows a fair agreement with that of the equilibrium intermediate. These results indicate that the molten globule state of equine beta-lactoglobulin populates at moderate urea concentration as well as at acid pH and it is equivalent with the kinetic folding intermediate.