Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hy-drochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteol-ysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conforma-tional change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0.75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at th     

Reversible thermal denaturation of human FGF-1 induced by low concentrations of guanidine hydrochloride.

Human acidic fibroblast growth factor (FGF-1) is a powerful mitogen and angiogenic factor with an apparent melting temperature (Tm) in the physiological range. FGF-1 is an example of a protein that is regulated, in part, by stability-based mechanisms. For example, the low Tm of FGF-1 has been postulated to play an important role in the unusual endoplasmic reticulum-independent secretion of this growth factor. Despite the close relationship between function and stability, accurate thermodynamic parameters of unfolding for FGF-1 have been unavailable, presumably due to effects of irreversible thermal denaturation. Here we report the determination of thermodynamic parameters of unfolding (DeltaH, DeltaG, and DeltaCp) for FGF-1 using differential scanning calorimetry (DSC). The thermal denaturation is demonstrated to be two-state and reversible upon the addition of low concentrations of added guanidine hydrochloride (GuHCl). DeltaG values from the DSC studies are in excellent agreement with values from isothermal GuHCl denaturation monitored by fluorescence and circular dichroism (CD) spectroscopy. Furthermore, the results indicate that irreversible denaturation is closely associated with the formation of an unfolding intermediate. GuHCl appears to promote reversible two-state denaturation by initially preventing aggregation of this unfolding intermediate, and at subsequently higher concentrations, by preventing formation of the intermediate.     

The calculation of partial specific volumes of proteins in guanidine hydrochloride

The effects of various modifiers on the ATPase activity of bovine platelet actomyosin has been studied. The order of activation by monovalent cations was NH₄⁺? K⁺ > Li⁺ > Na⁺. The order of activation by divalent cations was Ca²⁺ > Mn²⁺ = Sr²⁺ > Ba²⁺> Co²⁺ > Mg²⁺ > Zn²⁺. Ethylenediaminetetraacetate inhibits. Activity increased with increasing concentrations of monovalent cations, except for inhibition by increasing concentrations of NH₄⁺ in the presence of Ca²⁺. Adenosine triphosphatase activity was increased by low concentrations of urea and trypsin, but was unaffected by low concentrations of N-ethylmaleimide. For all enzymatic properties where direct comparisons are possible, actomyosin from platelets is unlike that from skeletal muscle, but is similar to that from smooth muscle and non-muscle sources.     

Extended conformations of polypeptides and proteins in urea and guanidine hydrochloride

By analyzing the effect of urea and guanidine hydrochloride on the circular dichroism of many polypeptides and proteins, it is concluded that under conditions of high concentration of the perturbant and at low temperatures the resultant state approached is that of a local extended helix structure instead of a completely random coil. Intensification by urea and guanidine hydrochloride of the circular dichroism bands of poly-L -proline II leads to the proof that the mechanism of interaction of urea and guanidine hydrochloride with proteins is through hydrogen bonding to the backbone carbonyl group.     

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.     

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.     

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

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

Conformational transitions of thioredoxin in guanidine hydrochloride

Spectral and hydrodynamic measurements of thioredoxin from Escherichia coli indicate that the compact globular structure of the native protein is significantly unfolded in the presence of guanidine hydrochloride concentrations in excess of 3.3 M at neutral pH and 25 degrees C. This conformational transition having a midpoint at 2.5 M denaturant is quantitatively reversible and highly cooperative. Stopped-flow measurements of unfolding in 4 M denaturant, observed with tryptophan fluorescence as the spectral probe, reveal a single kinetic phase having a relaxation time of 7.1 +/- 0.2 s. Refolding measurements in 2 M denaturant reveal three kinetic phases having relaxation times of 0.54 +/- 0.23, 14 +/- 6, and 500 +/- 130 s, accounting for 12 +/- 2%, 10 +/- 1%, and 78 +/- 3% of the observed change in tryptophan fluorescence. The dominant slowest phase is generated in the denatured state with a relaxation time of 42 s observed in 4 M denaturant. Both the slowest phase observed in refolding and the generation of the slowest phase in the denatured state have an activation enthalpy of 22 +/- 1 kcal/mol. These features of the slowest phase are compatible with an obligatory peptide isomerization of proline-76 to its cis isomer prior to refolding.     

Charge-transfer studies of the availability of aromatic side chains of proteins in guanidine hydrochloride.

The ability of aromatic tryptophyl and tyrosyl side-chain donors to form charge-transfer (CT) complexes with the acceptor 1-methyl-3-carbamidopyridinium chloride has been used to investigate the degree of exposure of these aromatic residues in denaturated proteins. The coplanar geometry of the CT complexes requires that virtually a full ring face of the donor be available for interaction with the acceptor, and the aromatic donor residues of lysozyme, trypsin, chymotrypsin, and the zymogens of the latter two enzymes do not appear to be wholly "exposed" in 6 M guanidine hydrochloride. Comparison of the CT proerties of the proteins with the corresponding properties of model complexes suggests that the incomplete exposure is due at least in part to statistical fluctuations in the continuously mobile, randomly coiled polypeptide chain which result in residues being alternately fully exposed and partly covered. Reduction and alkylation of the disulfide cross-links increase the apparent availability of the aromatic residues but the exposure is still less than that expected from a comparable mixture of tryptophan and tyrosine residues. Previous studies on the exposure of the aromatic residues of lysozyme and trypsin in aqueous salt solutions, when taken together with the present results, further suggest that there are two distinct kinds of surface environment possible on native proteins in solution. Some residues appear to be located in areas of the protein surface which are characterized by relatively fixed or stable local conformations, and have apparent CT association constants closely resembling these of comparable model complexes. Other residues may be located in a region where the protein conformation is flexible or continuously mobile, as evidenced by their smaller apparent association constants. It is probably significant that Trp-62 of lysozyme and Trp-215 of trypsin, both specificity site residues, appear to belong to the class of residues which can be considered as being in a flexible environment on the protein surface.     

Molten globule intermediates of human serum albumin in low concentration of urea

Interaction of non-electrolytes such as urea with proteins especially at lower concentrations is opening-up newer concepts in the understanding of protein stability and folding in proteomics. In this study, the secondary and tertiary structural characteristics and thermal stability of human serum albumin at lower concentrations of urea have been monitored. The protein attains a molten globule like structure at concentration urea below 2 M. This structural state also shows an increase in the alpha-helical content as compared to the native state. At concentrations of urea above 2 M, human serum albumin starts unfolding, resulting in a three-state transition with two mid points of transitions at around 4 M and 7 M urea concentrations. The characteristics of the partially folded intermediates are discussed with respect to the three component system analyses. Preferential hydration dominates over preferential interaction at lower concentration of urea (up to 2.5 M) and at higher concentration, the preferential interaction overtakes preferential hydration in a competitive manner. Formation of structural intermediates at lower concentration of urea is hypothesized as a general phenomenon in proteins and fits in with the observation with preferential interaction parameters by Timasheff and co-workers in the case of lysozyme and ribonuclease at different pH values.     

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.