Alpha-crystallin and ATP facilitate the in vitro renaturation of xylanase: enhancement of refolding by metal ions

Alpha-crystallin is a multimeric protein that functions as a molecular chaperone and shares extensive structural homology to small heat shock proteins. For the functional in vitro analysis of alpha-crystallin, the xylanase Xyl II from alkalophilic thermophilic Bacillus was used as a model system. The mechanism of chaperone action of alpha-crystallin is less investigated. Here we studied the refolding of Gdn HCl-denatured Xyl II in the presence and absence of alpha-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1-naphthalene sulfonate binding studies, suggest that alpha-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of Xyl II. The alpha-crystallin.Xyl II complex exhibited no functional activity. Addition of ATP to the complex initiated the renaturation of Xyl II with 30%-35% recovery of activity. The nonhydrolyzable analog 5'-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active Xyl II to a lesser extent than ATP. Although the presence of Ca(2+) was not required for the in vitro refolding of Xyl II, the renaturation yield was enhanced in its presence. Experimental evidence indicated that the binding of ATP to the alpha-crystallin.Xyl II complex brought about conformational changes in alpha-crystallin facilitating the dissociation of xylanase molecules. This is the first report of the enhancement of alpha-crystallin chaperone functions by metal ions.     

Intermediates in the refolding of ribonuclease at subzero temperatures. 3. Multiple folding pathways

The kinetics of refolding of ribonuclease A have been measured at -15 degrees C by monitoring the intrinsic fluorescence and absorbance signals from the six tyrosine residues. For each probe multiphasic kinetics were observed. The burial of tyrosine residues, as determined by the change in absorbance at 286 nm, revealed four phases, whereas the kinetics of refolding monitored by fluorescence revealed only two phases. The rates of the transients detected by fluorescence were independent of pH. One of the faster transients detected by delta A286 involved a decrease in absorbance, which is consistent with solvent exposure, rather than burial, and suggests the possibility of an abortive partially folded intermediate in the earlier stages of folding. Double-jump unfolding assays were used to follow the buildup and decay of an intermediate in the refolding reaction at -15 degrees C. At both pH* 3.0 and pH* 6.0 the maximum concentration of the intermediate was 25-30% of the total protein. The existence of a second pathway of slow folding was inferred from the difference in rate of formation of native enzyme and breakdown of the observed intermediate, and by computer simulations. In addition, the unfolding assay demonstrated that 20% of the unfolded protein was converted to native at a much faster rate, consistent with observations in aqueous solution that 80% of unfolded ribonuclease A consists of slow-folding species. Kinetics and amplitude data from these and other refolding experiments with different probes were used to develop possible models for the pathway of refolding. The simplest system consistent with the results for the slow-refolding species involves two parallel pathways with multiple intermediates on each of them. Several independent lines of evidence indicate that about 30% of the unfolded state refolds by the minor pathway, in which the slowest observed phase is attributed to the isomerization of Pro-93. The major pathway involves 50% of the unfolded state; the reason why it refolds slowly is not apparent. A native-like intermediate is formed considerably more rapidly in the major slow-refolding pathway, compared to the minor pathway.     

Acid-induced partly folded conformation resembling a molten globule state of xylanase from an alkalothermophilic Bacillus sp.

Nonnative protein structures having a compact secondary, but not rigid tertiary structure, have been increasingly observed as intermediate states in protein folding. We have shown for the first time during acid-induced unfolding of xylanase (Xyl II) the presence of a partially structured intermediate form resembling a molten globule state. The conformation and stability of Xyl II at acidic pH was investigated by equilibrium unfolding methods. Using intrinsic fluorescence and CD spectroscopic studies, we have established that Xyl II at pH 1.8 (A-state) retains the helical secondary structure of the native protein at pH 7.0, while the tertiary interactions are much weaker. At variance, from the native species (N-state), Xyl II in the A-state binds 1-anilino-8-sulfonic acid (ANS) indicating a considerable exposure of aromatic side chains. Lower concentration of Gdn HCl are required to unfold the A-state. For denaturation by Gdn HCl, the midpoint of the cooperative unfolding transition measured by fluorescence for the N-state is 3.5 +/- 0.1 M, which is higher than the value (2.2 +/- 0.1 M) observed for the A-state at pH 1.8. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its structural stability that is characteristic for native proteins.     

Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase

The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.     

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.     

The mechanism of folding of globular proteins. Equilibria and kinetics of conformational transitions of penicillinase from Staphylococcus aureus involving a state of intermediate conformation.

1. The thermodynamically reversible unfolding and refolding of penicillinase between the native and fully unfolded states were followed by using guanidinium chloride as denaturant. 2. The equilibria, studied by optical rotation, u.v. absorption, viscosity and enzyme activity, show the presence of a state of intermediate conformation, termed state H, which is stable at 20 degrees C in 0.8 M-guanidinium chloride. 3. The physical properties of this state show that it is slightly expanded with an intrinsic viscosity of 8 ml-g-1, that the 13 tyrosine residues, which are distributed through the primary sequence, are maximally exposed to the solvent and that the helix content is the same as that of the native state. 4. The kinetics of the transition between the native state, state H and the fully unfolded state were followed by u.v. absorption and by optical rotation. They are interpreted as showing that state H lies on the folding pathway between the native and fully unfolded states. 5. The transition between the native state and state H exhibits monophasic unfolding kinetics and biphasic refolding kinetics. This indicates that there must be at least two intermediate states in this process, at least one of which lies on the folding pathway which may also involve cul-de-sac paths. 6. The results are discussed in terms of a mechanism involving rapid stabilization of nucleation regions in a moderately compact but internally solvated structure, with 'native format' [Anfinsen (1973) Science 181, 233-230] secondary structure stabilized by tertiary interaction. The final and rate-limiting step in refolding involves shuffling of these structural elements into the native state. 7. This model is discussed in relation to folding in vivo.     

Topology and sequence in the folding of a TIM barrel protein: global analysis highlights partitioning between transient off-pathway and stable on-pathway folding intermediates in the complex folding mechanism of a (betaalpha)8 barrel of unknown function from B. subtilis

The relative contributions of chain topology and amino acid sequence in directing the folding of a (betaalpha)(8) TIM barrel protein of unknown function encoded by the Bacillus subtilis iolI gene (IOLI) were assessed by reversible urea denaturation and a combination of circular dichroism, fluorescence and time-resolved fluorescence anisotropy spectroscopy. The equilibrium reaction for IOLI involves, in addition to the native and unfolded species, a stable intermediate with significant secondary structure and stability and self-associated forms of both the native and intermediate states. Global kinetic analysis revealed that the unfolded state partitions between an off-pathway refolding intermediate and the on-pathway equilibrium intermediate early in folding. Comparisons with the folding mechanisms of two other TIM barrel proteins, indole-3-glycerol phosphate synthase from the thermophile Sulfolobus solfataricus (sIGPS) and the alpha subunit of Escherichia coli tryptophan synthase (alphaTS), reveal striking similarities that argue for a dominant role of the topology in both early and late events in folding. Sequence-specific effects are apparent in the magnitudes of the relaxation times and relative stabilities, in the presence of additional monomeric folding intermediates for alphaTS and sIGPS and in rate-limiting proline isomerization reactions for alphaTS.     

A stable cold folding intermediate of rabbit muscle D-glyceraldehyde 3-phosphate dehydrogenase.

With decreasing temperature the reactivation yield of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) upon dilution increases but the reactivation rate decreases. Neither reactivation nor aggregation during refolding can be detected at 4 degrees C in 48 h, and at 3 degrees C even in 6 days. However, the reactivation takes place once the temperature is raised with little decrease of the yield after incubation for 6 days at 3 degrees C. A cold folding intermediate forms in a burst phase of refolding at 4 degrees C as shown by a fast change of the intrinsic fluorescence followed by further conformational adjustment to a stable state in about 1 h. The stable folding intermediate has been characterized to be a dimer of partially folded GAPDH subunit with secondary structure between that of the native and denatured enzymes, a hydrophobic cluster not found in either the native or the denatured state, and an active site similar to but different from that of the native state. Chaperonin 60 (GroEL) binds with all intermediates formed at 4 degrees C, but the intermediates formed at the early folding stage reactivate with higher yield than those formed after conformational adjustment when dissociated from GroEL in the presence of ATP and further folded and assembled into the native tetramer.     

Effects of the difference in the unfolded-state ensemble on the folding of Escherichia coli dihydrofolate reductase

The unfolded state of a protein is an ensemble of a large number of conformations ranging from fully extended to compact structures. To investigate the effects of the difference in the unfolded-state ensemble on protein folding, we have studied the structure, stability, and folding of "circular" dihydrofolate reductase (DHFR) from Escherichia coli in which the N and C-terminal regions are cross-linked by a disulfide bond, and compared the results with those of disulfide-reduced "linear" DHFR. Equilibrium studies by circular dichroism, difference absorption spectra, solution X-ray scattering, and size-exclusion chromatography show that whereas the native structures of both proteins are essentially the same, the unfolded state of circular DHFR adopts more compact conformations than the unfolded state of the linear form, even with the absence of secondary structure. Circular DHFR is more stable than linear DHFR, which may be due to the decrease in the conformational entropy of the unfolded state as a result of circularization. Kinetic refolding measurements by stopped-flow circular dichroism and fluorescence show that under the native conditions both proteins accumulate a burst-phase intermediate having the same structures and both fold by the same complex folding mechanism with the same folding rates. Thus, the effects of the difference in the unfolded state of circular and linear DHFRs on the refolding reaction are not observed after the formation of the intermediate. This suggests that for the proteins with close termini in the native structure, early compaction of a protein molecule to form a specific folding intermediate with the N and C-terminal regions in close proximity is a crucial event in folding. If there is an enhancement in the folding reflecting the reduction in the breadth of the unfolded-state ensemble for circular DHFR, this acceleration must occur in the sub-millisecond time-range.     

Folding of intracellular retinol and retinoic acid binding proteins

The folding mechanisms of cellular retinol binding protein II (CRBP II), cellular retinoic acid binding protein I (CRABP I), and cellular retinoic acid binding protein II (CRABP II) were examined. These beta-sheet proteins have very similar structures and higher sequence homologies than most proteins in this diverse family. They have similar stabilities and show completely reversible folding at equilibrium with urea as a denaturant. The unfolding kinetics of these proteins were monitored during folding and unfolding by circular dichroism (CD) and fluorescence. During unfolding, CRABP II showed no intermediates, CRABP I had an intermediate with nativelike secondary structure, and CRBP II had an intermediate that lacked secondary structure. The refolding kinetics of these proteins were more similar. Each protein showed a burst-phase change in intensity by both CD and fluorescence, followed by a single observed phase by both CD and fluorescence and one or two additional refolding phases by fluorescence. The fluorescence spectral properties of the intermediate states were similar and suggested a gradual increase in the amount of native tertiary structure present for each step in a sequential path. However, the rates of folding differed by as much as 3 orders of magnitude and were slower than those expected from the contact order and topology of these proteins. As such, proteins with the same final structure may not follow the same route to the native state.     

Equilibrium and kinetic studies on folding of canine milk lysozyme

Thermal and chemical unfolding studies of the calcium-binding canine lysozyme (CL) by fluorescence and circular dichroism spectroscopy show that, upon unfolding in the absence of calcium ions, a very stable equilibrium intermediate state is formed. At room temperature and pH 7.5, for example, a stable molten globule state is attained in 3 M GdnHCl. The existence of such a pure and stable intermediate state allowed us to extend classical stopped-flow fluorescence measurements that describe the transition from the native to the unfolded form, with kinetic experiments that monitor separately the transition from the unfolded to the intermediate state and from the intermediate to the native state, respectively. The overall refolding kinetics of apo-canine lysozyme are characterized by a significant drop in the fluorescence intensity during the dead time, followed by a monoexponential increase of the fluorescence with k = 3.6 s(-1). Furthermore, the results show that, unlike its drastic effect on the stability, Ca(2+)-binding only marginally affects the refolding kinetics. During the refolding process of apo-CL non-native interactions, comparable to those observed in hen egg white lysozyme, are revealed by a substantial quenching of tryptophan fluorescence. The dissection of the refolding process in two distinct steps shows that these non-native interactions only occur in the final stage of the refolding process in which the two domains match to form the native conformation.     

Folding mechanism of ketosteroid isomerase from Comamonas testosteroni

Ketosteroid isomerase (KSI) from Comamonas testosteroni is a homodimeric enzyme with 125 amino acids in each monomer catalyzing the allylic isomerization reaction at rates comparable to the diffusion limit. Kinetic analysis of KSI refolding has been carried out to understand its folding mechanism. The refolding process as monitored by fluorescence change revealed that the process consists of three steps with a unimolecular fast, a bimolecular intermediate, and most likely unimolecular slow phases. The fast refolding step might involve the formation of structured monomers with hydrophobic surfaces that seem to have a high binding capacity for the amphipathic dye 8-anilino-1-naphthalenesulfonate. During the refolding process, KSI also generated a state that can bind equilenin, a reaction intermediate analogue, at a very early stage. These observations suggest that the KSI folding might be driven by the formation of the apolar active-site cavity while exposing hydrophobic surfaces. Since the monomeric folding intermediate may contain more than 83% of the native secondary structures as revealed previously, it is nativelike taking on most of the properties of the native protein. Urea-dependence analysis of refolding revealed the existence of folding intermediates for both the intermediate and slow steps. These steps were accelerated by cyclophilin A, a prolyl isomerase, suggesting the involvement of a cis-trans isomerization as a rate-limiting step. Taken together, we suggest that KSI folds into a monomeric intermediate, which has nativelike secondary structure, an apolar active site, and exposed hydrophobic surface, followed by dimerization and prolyl isomerizations to complete the folding.     

Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.     

The refolding of type II shikimate kinase from Erwinia chrysanthemi after denaturation in urea.

Shikimate kinase was chosen as a convenient representative example of the subclass of alpha/beta proteins with which to examine the mechanism of protein folding. In this paper we report on the refolding of the enzyme after denaturation in urea. As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism (CD) and fluorescence, respectively, the enzyme was fully unfolded in 4 m urea. From an analysis of the unfolding curve in terms of the two-state model, the stability of the folded state could be estimated as 17 kJ.mol-1. Approximately 95% of the enzyme activity could be recovered on dilution of the urea from 4 to 0.36 m. The results of spectroscopic studies indicated that refolding occurred in at least four kinetic phases, the slowest of which (k = 0.009 s-1) corresponded with the regain of shikimate binding and of enzyme activity. The two most rapid phases were associated with a substantial increase in the binding of 8-anilino-1-naphthalenesulfonic acid with only modest changes in the far UV CD, indicating that a collapsed intermediate with only partial native secondary structure was formed rapidly. The relevance of the results to the folding of other alpha/beta domain proteins is discussed.     

Refolding intermediate of guanidine hydrochloride denatured aminoacylase

The refolding of aminoacylase denatured in 6M guanidine hydrochloride (GdnHCl) has been studied by measuring enzyme activity, fluorescence emission spectra, ANS fluorescence spectra and far-UV circular dichroism spectra. The results showed that GdnHCl-denatured aminoacylase could be refolded and reactivated by dilution. A refolding intermediate was observed for low concentrations of GdnHCl (between 0.5 and 1.2M). This refolding intermediate was characterized by an increased fluorescence emission intensity, a blue-shifted emission maximum, and by increased binding of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). The secondary structure of the intermediate was similar to that of the native enzyme, and was therefore quite similar to the molten globule state often found in the protein folding pathway. Combined with the previous evidence of existence of an intermediate during unfolding process, we therefore proposed that the unfolding and refolding of aminoacylase might share the same pathway. A comparison of the Apo-enzyme and Holo-enzyme showed that there was little effect of the zinc ion on the refolding of the aminoacylase. Our study, the first successful report of the refolding of this metalloenzyme, also showed that lowering the concentration and the temperature of the enzyme improved the refolding rate of aminoacylase. The system therefore provides a useful model to study the refolding of proteins with prosthetic groups.     

Unfolding pathway of CotA-laccase and the role of copper on the prevention of refolding through aggregation of the unfolded state

Copper is a redox-active metal and the main player in electron transfer reactions occurring in multicopper oxidases. The role of copper in the unfolding pathway and refolding of the multicopper oxidase CotA laccase in vitro was solved using double-jump stopped-flow experiments. Unfolding of apo- and holo-CotA was described as a three-state process with accumulation of an intermediate in between the native and unfolded state. Copper stabilizes the native holo-CotA but also the intermediate state showing that copper is still bound to this state. Also, copper binds to unfolded holo-CotA in a non-native coordination promoting CotA aggregation and preventing refolding to the native structure. These results gather information on unfolding/folding pathways of multicopper oxidases and show that copper incorporation in vivo should be a tight controlled process as copper binding to the unfolded state under native conditions promotes protein aggregation.     

The detection of kinetic intermediate(s) during refolding of rhodanese

Recent studies showed that the enzyme rhodanese could be reversibly unfolded in guanidinium chloride (GdmCl) if aggregation and oxidation were minimized. Further, these equilibrium studies suggested the presence of intermediate(s) during refolding (Tandon, S., and Horowitz, P. (1989) J. Biol. Chem. 264, 9859-9866). The present work shows that native and refolded enzymes are very similar in structural and functional characteristics. Kinetics of denaturation/renaturation were used to detect the folding intermediate(s). The shift in fluorescence wavelength maximum was used to monitor the structural changes during the process. First order plots of the structural changes during unfolding and refolding show nonlinear curves. The refolding occurs in at least two phases. The first phase is very fast (t1/2 much less than 30 s) and accounts for the partial regain in the structure but not in the activity. The second phase is slow (t1/2 = 2.9 h) during which the enzyme fully regains its structure along with the activity. The fractional renaturation of rhodanese due to the fast phase, monitored in various concentrations of GdmCl, describes a transition centered at 3.5 M GdmCl which is very similar to the higher of the two transitions observed in the reversible refolding. All of these findings support the presence of detectable intermediate(s) during folding of rhodanese.     

Folding/Unfolding Kinetics of Apomyoglobin

The equilibrium and kinetic folding/unfolding of apomyoglobin (ApoMb) were studied at pH 6.2, 11 °C by recording tryptophan fluorescence. The equilibrium unfolding of ApoMb in the presence of urea was shown to involve accumulation of an intermediate state, which had a higher fluorescence intensity as compared with the native and unfolded states. The folding proceeded through two kinetic phases, a rapid transition from the unfolded to the intermediate state and a slow transition from the intermediate to the native state. The accumulation of the kinetic intermediate state was observed in a wide range of urea concentrations. The intermediate was detected even in the region corresponding to the unfolding limb of the chevron plot. Urea concentration dependence was obtained for the observed folding/unfolding rate. The shape of the dependence was compared with that of two-state proteins characterized by a direct transition from the unfolded to the native state.     

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.     

Temperature control for kinetic refolding of heat-denatured ovalbumin.

The folding of heat-denatured ovalbumin, a non-inhibitory serpin with a molecular size of 45 kDa, was examined. Ovalbumin was heat-denatured at 80 degrees C under nonreducing conditions at pH 7.5 and then cooled either slowly or rapidly. Slow cooling allowed the heat-denatured ovalbumin to refold to its native structure with subsequent resistance to digestion by trypsin. Upon rapid cooling, by contrast, the heat-denatured molecules assumed the metastable non-native conformations that were susceptible to trypsin. The non-native species were marginally stable for several days at a low temperature, but the molecules were transformed slowly into the native conformation. Considering data from size-exclusion chromatography and from analyses of CD, intrinsic tryptophan fluorescence, and adsorption of the dye 1-anilinonaphthalene-8-sulfonate, we postulated that the non-native species that accumulated upon rapid cooling were compact but structureless globules with disordered side chains collectively as a folding intermediate. Temperature-jumped CD experiments revealed biphasic kinetics for the refolding process of heat-denatured ovalbumin, with the features of increasing and subsequently decreasing amplitude of the rapid and the slow phases, respectively, with the decrease in folding temperature. The temperature dependence of the refolding kinetics indicated that the yield of renaturation was maximal at about 55 degrees C. These findings suggested the kinetic partitioning of heat-denatured ovalbumin between alternative fates, slow renaturation to the native state and rapid collapse to the metastable intermediate state. Analysis of disulfide pairing revealed the formation of a scrambled form with non-native disulfide interactions in both the heat-denatured state and the intermediate state that accumulated upon rapid cooling, suggesting that non-native disulfide pairing is responsible for the kinetic barriers that retard the correct folding of ovalbumin.