Aggregation, dissociation and unfolding of glucose dehydrogenase during urea denaturation

The effect of urea on glucose dehydrogenase from Bacillus megaterium has been studied by following changes in enzymatic activity, conformation and state of aggregation. It was found that the denaturation process involves several transitions. At very low urea concentrations (below 0.5 M), where the enzyme is fully active and tetrameric, there is a conformational change as monitored by an increase in intensity of the tryptophan fluorescence and a maximum exposure of organized hydrophobic surfaces as reported by the fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5.5'-disulfonic acid. At slightly higher urea concentrations (0.75-2 M), a major conformational transition occurs, as monitored by circular dichroism and fluorescence measurements, in which the enzyme activity is completely lost and is concomitant with the formation of interacting intermediates that lead to a highly aggregated state. Increasing urea concentrations cause a complete dissociation to lead first a partially and eventually the complete unfolded monomer. These phenomena are fully reversible by dilution of denaturant. It is concluded that after urea denaturation, the folding/assembly pathway of glucose dehydrogenase occurs with the formation of intermediate species in which transient higher aggregates appear to be involved.     

Unfolding and inactivation of Ampullarium crossean beta-glucosidase during denaturation by guanidine hydrochloride

Changes of activity and conformation of Ampullarium crossean beta-glucosidase in different concentrations of guanidine hydrochloride (GuHCl) have been studied by measuring the fluorescence spectra and its relative activity after denaturation. The fluorescence intensity of the enzyme decreased distinctly with increasing guanidine concentrations, the emission peaks appeared red shifted (from 338.4 to 350.8 nm), whereas a new fluorescence emission peak appeared near 310 nm. Changes in the conformation and catalytic activity of the enzyme were compared. A corresponding rapid decrease in catalytic activity of the enzyme was also observed. The extent of inactivation was greater than that of conformational changes, indicating that the active site of the enzyme is more flexible than the whole enzyme molecule. k(+0)>k(+0)' also showed that the enzyme was protected by substrate to a certain extent during guanidine denaturation.     

Dissociation and aggregation of D-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride

The inactivation of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) (GAPDH) during guanidine hydrochloride (GdnHCl) denaturation has been compared with its state of aggregation and unfolding, by light scattering and fluorescence measurements. The enzyme first dissociates at low concentrations of GdnHCl, followed by the formation of a highly aggregated state with increasing denaturant concentrations, and eventually by complete unfolding and dissociation to the monomer at concentrations of greater than 2 M GdnHCl. The aggregation and final dissociation correspond roughly with the two stages of fluorescence changes reported previously (Xie, G.-F. and Tsou, C.-L. (1987) Biochim. Biophys. Acta 911, 19-24). Rate measurements show a very rapid inactivation, the extents of which increase with increasing concentrations of GdnHCl. This initial rapid phase of inactivation which takes place before dissociation and unfolding of the molecule is in agreement with the results obtained with other enzymes, that the active site is affected before noticeable conformational changes can be detected for the enzyme molecule as a whole. A scheme for the steps leading to the final denaturation, and dissociation of the enzyme to the inactive and unfolded monomer, is proposed.     

Identification and characterization of functional intermediates of stem bromelain during urea and guanidine hydrochloride unfolding

By comparing changes in enzyme activity with changes in spectral features for stem bromelain (EC.3.4.22.32) in the absence and presence of urea, Guanidine hydrochloride and ethanol; four intermediate states could be identified: two activity-enhanced state obtained in the presence of 5 M urea and 2 M GnHCl, termed X and X', respectively, and a third, similarly active state closely resembling the native protein in the presence of 8-9 M urea, termed Y. The enhanced activity of these states is due to local conformational changes accompanied by increased dynamics in the active site. Further, the enzyme does not lose its activity after substantial tertiary structure changes in 8-9 M urea (Y state), suggesting that active site containing domain is more resistant to chemical denaturation than the other structural domain. This makes stem bromelain and in general cysteine proteases an exception to the hypothesis that active site is the most labile part of enzyme.     

Evidence for the existence of an unfolding intermediate of thyroglobulin during denaturation by guanidine hydrochloride

The unfolding of bovine thyroglobulin (Tg) in guanidine hydrochloride (GuHCl) solution was studied by following the fluorescence and circular dichroism. With increasing GuHCl concentrations, the emission maximum of the intrinsic fluorescence clearly red-shifted in two stages. At concentrations of GuHCl less than 1.2 M or more than 1.6 M, the red shift showed a cooperative manner. At concentrations of GuHCl between 1.2 and 1.6 M, an unfolding intermediate was observed. It was further characterized by the increased binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid (ANS). No significant changes of the secondary structure were indicated by CD spectra at the concentrations of GuHCl between 1.2 and 1.6 M. The conformation of this state has properties similar to those of a molten globule state which may exist in the folding pathway of the protein. Further changes in fluorescence properties occurred at concentrations of denaturant higher than 1.6 M with a significant red shift of the emission maximum from 340 to 347 nm and a marked decrease in ANS binding. This in vitro study gave a clue to understand the biochemical mechanism for the occurrence of aggregation and molecular chaperone binding during Tg maturation in vivo.     

Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K_m values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 ± 0.2 × 10⁻⁸ mol L⁻¹, 1.4 ± 0.2 × 10⁻⁸ mol L⁻¹, and 1.6 ± 0.2 × 10⁻⁸ mol L⁻¹, respectively. The dissociation constant (K_d) showed changes in the affinity of the enzyme for the substrate with increases in the K_cat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.     

Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation

Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m--the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration--these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified--interactions which appear to involve hydrophobic groups.     

pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1

To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.     

Comparison of the stability of fish light meromyosins by guanidine hydrochloride denaturation

The guanidine hydrochloride denaturation of light meromyosins (LMMs) of fish (carp, sardine and greenling) and rabbit was investigated to determine their structural stability quantitatively. The circular dichroism (CD) and fluorescence spectroscopies were applied to monitor denaturation. The CD results indicate that the LMM α-helix undergoes a two-step unfolding. The free energy of denaturation was calculated based on the linear extrapolation method and the denaturant binding model. Total free energies of the two-step unfolding of the α-helix are related to the water temperatures in which the fish live and the body temperature of rabbit. The stability of α-helical structure of LMM was in the following descending order: rabbit>carp>sardine>greenling. The free energies of denaturation obtained by tryptophan fluorescence differ from the free energies of the unfolding α-helix. The data from the two spectroscopic measurements are discussed along with the conformational changes of LMMs.     

Comparison of the rates of inactivation and conformational changes of creatine kinase during urea denaturation

The denaturation of creatine kinase in urea solutions of different concentrations has been studied by following the changes in the ultraviolet absorbance and intrinsic fluorescence as well as by the exposure of hidden SH groups. In concentrated urea solutions, the denaturation of the enzyme results in negative peaks at 285 nm with shoulders at 280 and 290 nm and positive peaks at 244 and 302 nm in the denatured minus native enzyme difference spectrum. The fluorescence emission maximum of the enzyme red shifts with increasing intensity in urea solutions of increasing concentrations. At least part of these changes can be attributed to direct effects of urea on the exposed Tyr and Trp residues as shown by experiments with model compounds. The inactivation of this enzyme has been followed and compared with the conformational changes observed during urea denaturation. A marked decrease in enzyme activity is already evident at low urea concentrations before significant conformational changes can be detected by the exposure of hidden SH groups or by ultraviolet absorbance and fluorescence changes. At higher urea concentrations, the enzyme is inactivated at rates 3 orders of magnitude faster than the rates of conformational changes. The above results are in accord with those reported previously for guanidine denaturation of this enzyme [Yao, Q., Hou, L., Zhou, H., & Tsou, C.-L. (1982) Sci. Sin. (Engl. Ed.) 25, 1186-1193] and can best be explained by assuming that the active site of this enzyme is situated near the surface of the enzyme molecule and is sensitive to very slight conformational changes.     

Comparison of inactivation and conformational changes of D-glyceraldehyde-3-phosphate dehydrogenase during thermal denaturation

The inactivation of D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) EC 1.2.1.12) (GAPDH) during thermal denaturation has been compared to its dissociation-aggregation measured by light scattering and changes in secondary structure measured by CD in the far ultraviolet. The inactivation at 38.5 degrees C consists of two stages. The rate of the first stage is too fast to be followed by conventional methods. The extent of this fast stage inactivation increases with increasing temperature and, more markedly, with increasing pH. At this stage, the inactivation is reversible and no appreciable dissociation or change in secondary structure can be detected. The secondary structure of the enzyme is relatively heat stable, showing no appreciable change at 38.5 degrees C. At this temperature, the enzyme first dissociates within several minutes probably into dimers and with prolonged heating, it becomes irreversibly aggregated. The above results are in accord with the earlier suggestion, based on results obtained during denaturation of a number of enzymes by guanidine hydrochloride (GdnHCl) and urea, that for some enzymes the active site is situated in a region more susceptible to perturbation than the molecule as a whole (Tsou, C.-L. (1986) Trends Biochem. Sci. 11, 427).     

The unfolding intermediate state of calf intestinal alkaline phosphatase during denaturation in guanidine solutions

The equilibrium unfolding of calf intestinal alkaline phosphatase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and ultraviolet difference spectra. At low concentrations of GdmCl (< 1.6 M), the fluorescence intensity decreased with a slight red shift of the emission maximum from 332 nm to 344 nm. An unfolding intermediate state was observed at a broad concentration range of GdmCl as a denaturant (between 1.6 and 2.6 M). This intermediate was characterized by increased fluorescence emission intensity, ultraviolet difference absorption at 236 nm and 260 nm, as well as increased binding to the protein and red shift of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid.     

Conformational and activity changes during guanidine denaturation of D-glyceraldehyde-3-phosphate dehydrogenase

Changes in intrinsic protein fluorescence of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been compared with inactivation of the enzyme during denaturation in guanidine solutions. The holoenzyme is completely inactivated at guanidine concentrations less than 0.5 M and this is accompanied by a red shift of the emission maximum at 335 nm and a marked decrease in intensity of the intrinsic fluorescence. At 0.5 M guanidine, the inactivation is a slow process, with a first-order rate constant of 2.4 X 10(-3) s-1. A further red shift in the emission maximum and a decrease in intensity occur at guanidine concentrations higher than 1.5 M. The emission peak at 410 nm of the fluorescent NAD derivative introduced at the active site of this enzyme (Tsou, C.L. et al. (1983) Biochem. Soc. Trans. 11, 425-429) shows both a red shift and a marked decrease in intensity at the same guanidine concentration required to bring about the inactivation and the initial changes in the intrinsic fluorescence of the holoenzyme. It appears that treatment by low guanidine concentrations leads to both complete inactivation and perturbation of the active site conformation and that a tryptophan residue is situated at or near the active site.     

Protein stabilization by urea and guanidine hydrochloride

The urea, guanidine hydrochloride, salt, and temperature dependence of the rate of dissociation of CO from a nonequilibrium state of CO-bound native ferrocytochrome c has been studied at pH 7. The heme iron of ferrocytochrome c in the presence of denaturing concentrations of guanidine hydrochloride (GdnHCl) and urea prepared in 0.1 M phosphate, pH 7, binds CO. When the unfolded protein solution is diluted 101-fold into CO-free folding buffer, the protein chain refolds completely, leaving the CO molecule bonded to the heme iron. Subsequently, slow thermal dissociation of the CO molecule yields to the heme coordination of the native M80 ligand. Thus, the reaction monitors the rate of thermal conversion of the CO-liganded native ferrocytochrome c to the M80-liganded native protein. The rate of this reaction, k(diss), shows a characteristic dependence on the presence of nondenaturing concentrations of the denaturants in the reaction medium. The rate decreases by approximately 1.9-3-fold as the concentration of GdnHCl in the refolding medium increases from nearly 0 to approximately 2.1 M. Similarly, the rate decreases by 1.8-fold as the urea concentration is raised from 0.l to approximately 5 M. At still higher concentrations of the denaturants the denaturing effect sets in, the protein is destabilized, and hence the CO dissociation rate increases sharply. The activation energy of the reaction, E(a), increases when the denaturant concentration in the reaction medium is raised: from 24.1 to 28.3 kcal mol(-1) for a 0.05-2.1 M rise in GdnHCl and from 25.2 to 26.9 kcal mol(-1) for a 0.1-26.9 M increase in urea. Corresponding to these increases in denaturant concentrations are also increases in the activation entropy, S(diss)/R, where R is the gas constant of the reaction. The denaturant dependence of these kinetic and thermodynamic parameters of the CO dissociation reaction suggests that binding interactions with GdnHCl and urea can increase the structural and energetic stability of ferrocytochrome c up to the limit of the subdenaturing concentrations of the additives. NaCl and Na(2)SO(4), which stabilize proteins through their salting-in effect, also decrease the rate with a corresponding increase in activation entropy of CO dissociation from CO-bound native ferrocytochrome c, lending support to the view that low concentrations of GdnHCl and urea stabilize proteins. These results have direct relevance to the understanding and interpretation of the free energy-denaturant relationship and protein folding chevrons.     

Differences in the unfolding of procerain induced by pH, guanidine hydrochloride, urea, and temperature

The structural and functional aspects along with equilibrium unfolding of procerain, a cysteine protease from Calotropis procera, were studied in solution. The energetic parameters and conformational stability of procerain in different states were also estimated and interpreted. Procerain belongs to the alpha + beta class of proteins. At pH 2.0, procerain exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is predominantly a beta-sheet conformation and exhibits strong ANS binding. GuHCl and temperature denaturation of procerain in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts in the molecular structure of procerain, possibly domains, with different stability that unfolds in steps. Moreover, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. At lower pH, procerain unfolds to the acid-unfolded state, and a further decrease in the pH drives the protein to the A state. The presence of 0.5 M salt in the solvent composition directs the transition to the A state while bypassing the acid-unfolded state. GuHCl-induced unfolding of procerain at pH 3.0 seen by various methods is cooperative, but the transitions are noncoincidental. Besides, a strong ANS binding to the protein is observed at low concentrations of GuHCl, indicating the presence of an intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8 M), procerain retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to unfolding by urea at lower pH, and the transitions are cooperative and coincidental. Further, the properties of the molten globule-like state and the intermediate state are different, but both states have the same conformational stability. This indicates that these intermediates may be located on parallel folding routes of procerain.     

Three-state denaturation of alpha-lactalbumin by guanidine hydrochloride.

The reversible unfolding of α-lactalbumin by guanidine hydrochloride has been studied at 25.0 °C by means of ultraviolet circular dichroism measurements. The non-coincidence of the apparent transition curves obtained from the ellipticity changes at far (222 nm) and at near (270 nm and 296 nm) ultraviolet wave-lengths demonstrates the presence of at least one intermediate in the denaturation process. The aromatic residues which contribute to the Cotton effects at 270 nm and at 296 nm appear to be exposed to solvent in the first stage of a two-stage process, while the helical regions of the polypeptide chain appear to be destroyed in the second stage. Earlier work has demonstrated an acid transition between two compact forms of α-lactalbumin, a native (neutral pH) form and an acid form. Results presented here suggest that the acid form is produced as an intermediate in the first stage of total unfolding at neutral pH.Lysozyme and α-lactalbumin are known to have similar primary structures and are expected to have similar tertiary structures, but several differences in their properties have been described. The comparison of the unfolding transitions of α-lactalbumin and lysozyme provides a result compatible with similar tertiary structures, although the free energy of stabilization of the native state is 3 to 5 kcal/mol smaller for α-lactalbumin than for lysozyme. The pH dependence of the unfolding reaction can be described in terms of abnormal histidyl and carboxyl residues. The presence of a stable intermediate in the denaturation process may cause a difference in dynamic character in the native state between the two proteins and thus provide a reasonable interpretation for their known differences in chemical reactivity.     

Refolding and reactivation of liver alcohol dehydrogenase after dissociation and denaturation in 6M guanidine hydrochloride.

Horse-liver alcohol dehydrogenase has been dissociated and denatured by 6 M guanidinium hydrochloride. Removal of the denaturant under optimum conditions of the solvent leads to partial reactivation. The concentrations of the enzyme, as well as the coenzyme (NAD+), and Zn2+, affect the reactivation significantly, since high concentrations promote the formation of inactive aggregation products. Analyzing the kinetics of reactivation and reassociation, conditions far from equilibrium of dissociation-association provide maximum yields (approximately 70%). The sigmoidal kinetic traces suggest a superposition of first-order transconformation and second-order association reactions; the latter are corroborated by the concentration dependence of the reactivation reaction. The coenzyme, NAD+, has no influence on the kinetics of reactivation. Addition of Zn2+ leads to a significant decrease of the rate and yield of reactivation. The process of renaturation, as reflected by the regain of native fluorescence shows complex kinetics: rapid relaxations are followed by slower first-order and second-order processes which parallel reactivation.     

Domain unfolding and the stability of thermolysin in guanidine hydrochloride

Equilibrium and kinetic studies of the unfolding and autolysis of the two domain protein thermolysin in guanidine hydrochloride are described. Enzyme activity, circular dichroism, fluorescence, sedimentation, size exclusion chromatography, and viscosity measurements were used to monitor conformational transitions and characterize the native and denatured states. The observation of biphasic transitions for the unfolding of apothermolysin and the spectroscopic changes associated with each phase of the overall unfolding process suggest unfolding of the N-terminal domain at less than 1 M guanidine hydrochloride, followed by the unfolding of the C-terminal domain, with the transition midpoint at 3 M guanidine hydrochloride. The refolding of the C-terminal domain is reversible; however, refolding of the N-terminal domain could not be demonstrated owing to protein aggregation. A quantitative analysis of the two transitions suggest that the unfolding of the two structural domains of thermolysin is not completely independent. Attempts to measure the unfolding of holothermolysin were hampered by autolysis. However, it was possible to show that at least three calcium ions serve to stabilize thermolysin against autolysis or unfolding in guanidine hydrochloride. Similar stabilization was observed for thermolysin with a single terbium ion bound at calcium site S(1). This result is consistent with our earlier findings, which suggest that calcium bound at sites S(1)-S(2) are located at a critical point on the unfolding pathway of thermolysin and serve to act as an interdomain lock.     

Free energy changes in ribonuclease A denaturation. Effect of urea, guanidine hydrochloride, and lithium salts

The unfolding of ribonuclease A by urea, guanidine hydrochloride, lithium perchlorate, lithium chloride, and lithium bromide has been followed by circular dichroic and difference spectral measurements. All three abnormal tyrosyl residues are normalized in urea and guanidine hydrochloride (delta epsilon 287 = -2700), only two are normalized in lithium bromide and lithium perchlorate (delta epsilon 287 = -1700), and only one is exposed in lithium chloride solutions (delta epsilon 287 = -700). The Gibbs energies are 4.7 +/- 0.1 kcal mol-1 for urea- and guanidine hydrochloride-denaturation, 3.8 +/- 0.2 kcal mol-1 for lithium perchlorate-denaturation, and 12.7 +/- 0.2 kcal mol-1 for lithium chloride- and lithium bromide-denaturation of ribonuclease A. The latter results suggest that the mechanism of the unfolding process in urea and guanidine hydrochloride is quite different from that in lithium salts.