Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl),pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65°C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasingpH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55°C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5°C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.     

The denaturation of circular enterocin AS-48 by urea and guanidinium hydrochloride

The unfolding thermodynamics of the circular enterocin protein AS-48, produced by Enterococcus faecalis, has been studied. The native structure of the 70-amino-acid-long protein turned out to be extremely stable against heat and denaturant-induced unfolding. At pH 2.5 and low ionic strength, it denatures at 102 degrees C, while at 25 degrees C, the structure only unfolds in 6.3 M guanidinium hydrochloride (GuHCl) and does not unfold even in 8 M urea. A comparison of its thermal unfolding in water and in the presence of urea shows a good correspondence between the two deltaGw(298) values, which are about 30 kJ mol(-1) at pH 2.5 and low ionic strength. The stability of the structure is highly dependent upon ionic strength and so GuHCl acts both as a denaturant and a stabilising agent. This seems to be why the deltaGw(298) value calculated from the unfolding data in GuHCl is twice as high as in the absence of this salt. At least part of the high stability of native AS-48 can almost certainly be put down to its circular organization since other structural features are quite normal for a protein of this size.     

The denaturation of rabbit muscle phosphorylase b by guanidinium chloride.

The denaturation of phosphorylase b by guanidinium chloride (GdnHCl) was studied. The enzyme is unusually sensitive to the denaturing agent, being more than 50% inactivated after incubation for 15 min in 0.1 M-GdnHCl. Full activity can be regained on dilution of the GdnHCl to 0.01 M, provided that the initial concentration of GdnHCl is less than 0.5 M. Studies of protein fluorescence, thiol-group reactivity, circular dichroism and absorption spectroscopy indicate that phosphorylase b undergoes slow structural changes in the range of GdnHCl concentrations from 0.5 to 0.8 M. The enzyme retains considerable folded structure even after 15 min incubation in 1 M-GdnHCl, but is rapidly and completely unfolded in 3 M-GdnHCl.     

Dilatometric studies of the denaturation of immunoglobulin G by guanidinium chloride

The denaturation of immunoglobulin G and its light chains by guanidinium chloride at 25°C was followed using the dilatometric method. From results of the dilatometric measurements the differences between the partial molar volumes of the proteins in water and in guanidinium chloride solutions of various concentrations, respectively, have been obtained. The differences reflect the extent of unfolding as well as the denaturant binding to the protein. Several experiments were also performed in which the protein disulfide bonds were reduced by dithioerythritol.The volume change produced by the reduction of one mole of disulfide bonds of immunoglobulin G in 6 M guanidinium chloride was found to be the same as that for oxidized glutathione; the value was ?0.9 ml/mol.     

A new method for determining the constant-pressure heat capacity change associated with the protein denaturation induced by guanidinium chloride (or urea)

Differential scanning calorimetry (DSC) provides authentic and accurate value of DeltaC(p)(X), the constant-pressure heat capacity change associated with the N (native state)<-->X (heat denatured state), the heat-induced denaturation equilibrium of the protein in the absence of a chemical denaturant. If X retains native-like buried hydrophobic interaction, DeltaC(p)(X) must be less than DeltaC(p)(D), the constant-pressure heat capacity change associated with the transition, N<-->D, where the state D is not only more unfolded than X but it also has its all groups exposed to water. One problem is that for most proteins D is observed only in the presence of chemical denaturants such as guanidinium chloride (GdmCl) and urea. Another problem is that DSC cannot yield authentic DeltaC(p)(D), for its measurement invokes the existence of putative specific binding sites for the chemical denaturants on N and D. We have developed a non-calorimetric method for the measurements of DeltaC(p)(D), which uses thermodynamic data obtained from the isothermal GdmCl (or urea)-induced denaturation and heat-induced denaturation in the presence of the chemical denaturant concentration at which significant concentrations of both N and D exist. We show that for each of the proteins (ribonuclease-A, lysozyme, alpha-lactalbumin and chymotrypsinogen) DeltaC(p)(D) is significantly higher than DeltaC(p)(X). DeltaC(p)(D) of the protein is also compared with that estimated using the known heat capacities of amino acid residues and their fractional area exposed on denaturation.     

Thermodynamics of the isothermal denaturation of lysozyme by guanidinium chloride.

A thermodynamic analysis of the isothermal denaturation of lysozyme by guanidinium chloride has been performed. The analysis is based on the equation which relates the equilibrium constant for denaturation to the preferential binding of denaturant. The equation has been derived previously by thermodynamic methods, whereas in this article a derivation based on statistical mechanics is given. By application of the equation the free energy of denaturation is first calculated and from it, by subtracting the calorimetrically-determined enthalpy of denaturation, the entropy of denaturation is determined.     

Water stress induced alterations in ornithine aminotransferase of ragi (Eleusine coracana): Protection by proline against heat inactivation and denaturation by urea and guanidinium chloride

Water stress resulted in a specific response leading to a large and significant increase (80-fold) in free proline content of ragi (Eleusine coracana leaves and seedlings. L-Proline protected ornithine aminotransferase, an enzyme in the pathway for proline biosynthesis, isolated from normal and stressed ragi leaves against heat inactivation and denaturation by urea and guanidinium chloride. The protection of the stressed enzyme by L-proline was much more complete than that of the enzyme isolated from normal leaves. While L-ornithine, one of the substrates, protected the stressed enzyme against inactivation, it enhanced the rate of inactivation of the normal enzyme. α-Ketoglutarate protected both the normal and stressed enzyme against inactivation and denaturation. These results support the suggestion that ornithine aminotransferase has undergone a structural alteration during water stress. In view of the causal relationship between elevated temperature and water stress of plants under natural conditions, the protection afforded by proline against inactivation and denaturation of the enzyme from stressed leaves assumes significance. These results provide an explanation for a possible functional importance of proline accumulation during water stress.     

Biphasic denaturation of human placental alkaline phosphatase in guanidinium chloride

Human placental alkaline phosphatase is a membrane-anchored dimeric protein. Unfolding of the enzyme by guanidinium chloride (GdmCl) caused a decrease of the fluorescence intensity and a large red-shifting of the protein fluorescence maximum wavelength from 332 to 346 nm. The fluorescence changes were completely reversible upon dilution. GdmCl induced a clear biphasic fluorescence spectrum change, suggesting that a three-state unfolding mechanism with an intermediate state was involved in the denaturation process. The half unfolding GdmCl concentrations, [GdmCl]_0.5, corresponding to the two phases were 1.45 M and 2.50 M, respectively. NaCl did not cause the same effect as GdmCl, indicating that the GdmCl-induced biphasic denaturation is not a salt effect. The decrease in fluorescence intensity was monophasic, corresponding to the first phase of the denaturation process with [GdmCl]_0.5 = 1.37 M and reached a minimum at 1.5 M GdmCl, where the enzyme remained completely active. The enzymatic activity lost started at 2.0 M GdmCl and was monophasic but coincided with the second-phase denaturation with [GdmCl]_0.5 = 2.46 M. Inorganic phosphate provides substantial protection of the enzyme against GdmCl inactivation. Determining the molecular weight by sucrose-density gradient ultracentrifugation revealed that the enzyme gradually dissociates in both phases. Complete dissociation occurred at [GdmCl] > 3 M. The dissociated monomers reassociated to dimers after dilution of the GdmCl concentration. Refolding kinetics for the first-phase denaturation is first-order but not second-order. The biphasic phenomenon thereby was a mixed dissociation-denaturation process. A completely folded monomer never existed during the GdmCl denaturation. The biphasic denaturation curve thereby clearly demonstrates an enzymatically fully active intermediate state, which could represent an active-site structure intact and other structure domains partially melted intermediate state. Proteins 33:49–61, 1998. © 1998 Wiley-Liss, Inc.     

Conformational changes in gastric mucoproteins induced by caesium chloride and guanidinium chloride

1. Caesium chloride and guanidinium chloride were shown to cause conformational changes in the high-molecular-weight mucoprotein A of water-soluble gastric mucus with no change in molecular weight. 2. Increasing concentrations of CsCl decrease the viscosity of the mucoprotein bringing about a transition which is essentially complete in 0.1m-CsCl. The shear-dependence of viscosity of the mucoprotein is abolished by low concentrations of CsCl. The normally highly expanded molecule becomes contracted in CsCl to a molecule having the same symmetry but a smaller volume and decreased solvation, in keeping with an increased sedimentation coefficient (18.7S-->33S). 3. This contracted form does not revert to the native conformation on removal of the CsCl. 4. A mechanism is discussed in terms of the effect of the Cs(+) and Cl(-)ions on water structure and the water-mucoprotein interaction. 5. Guanidinium chloride causes the CsCl-treated material to expand, in keeping with a decrease in s(0) (25,w) (33S-->26S). This is analogous to the known unfolding effect of guanidinium chloride on proteins and suggests that guanidinium chloride solubilizes groups involved in stabilizing the contracted structure. Removal of the guanidinium chloride results in a limited aggregation of four mucoprotein molecules. 6. These results show that caution must be exercised before interpreting the physical properties of mucoproteins which have been treated with CsCl and/or guanidinium chloride.     

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.     

The effect of urea and guanidinium chloride on activity of subtilisin Carlsberg

As shown by viscosity and optical rotation dispersion measurements, subtilisin Carlsberg is not denatured in the presence of 10 M urea or 6 M guanidinium chloride. This unusual structural stability made it possible to investigate the effects of these hydrophobic-bond breaking solutes on various aspects of the enzymic interaction with substrates and inhibitors. The binding of the competitive inhibitor N-benzoylarginine was decreased by urea or guanidinium chloride. The nature of this effect was such as to implicate hydrophobic interaction as making a major contribution to the binding. By contrast, Ks for the substrates N-acetyltyrosine ethyl ester, N-benzoylarginine ethyl ester and N-trans-cinnamoylimidazole was apparently unchanged by the presence of urea or guanidinium chloride. The influence of these solutes on kcat for the substrates was rather involved. Tentative hypotheses are put forward to account for the effects seen.     

Optical properties and denaturation by guanidinium chloride and urea of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of four molecular forms of the enzyme.

1. The fluorescence and circular dichroism of four homogeneous preparations of ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus differing in molecular structure and enzymic properties were examined at pH 7.5 and 25 degrees. Emission was maximum at 325 and 335 nm and the relative intensities at these wavelengths may be used to characterize the different ATPase preparations. The circular-dichroism spectra exhibited negative extrema at 208 and 220 nm, and the relative value of the molar ellipticity at these wavelengths was also different for each molecular form of the enzyme. 2. The four preparations undergo two consecutive major unfolding transitions in guanidinium chloride (midpoints at 0.94 and 1.5 M denaturant), with concomitant destruction of the quaternary structure of the protein. A comparatively minor alteration in the ATPase structure also occurred in 0.05-0.2M-guanidine and led to complete inactivation of the enzyme. The inactivation and the first unfolding transition were reversible by dilution of the denaturant; the transition with midpoint at 1.5M-guanidine was irreversible. 3. Similar results were obtained in urea, except that the successive transitions had midpoints at concentrations of denaturant of 0.4, 2.0 and 4.5M. Low concentrations of urea caused a noticeable activation of the enzyme activity and alterations of the electrophoretic mobility of the ATPase. 4. A model is proposed in which one of the major subunits, alpha, is first dissociated and unfolded reversibly by the denaturants, followed by the irreversible unfolding and dissociation of the other major subunit, beta, from subunit delta and/or the components of relative mobility 1.0 in dodecyl sulphate/polyacrylamide-gel electrophoresis (rho).     

Protein interactions with urea and guanidinium chloride. A calorimetric study.

The interaction of urea and guanidinium chloride with proteins has been studied calorimetrically by titrating protein solutions with denaturants at various fixed temperatures, and by scanning them with temperature at various fixed concentrations of denaturants. It has been shown that the observed heat effects can be described in terms of a simple binding model with independent and similar binding sites. Using the calorimetric data, the number of apparent binding sites for urea and guanidinium chloride have been estimated for three proteins in their unfolded and native states (ribonuclease A, hen egg white lysozyme and cytochrome c). The intrinsic and total thermodynamic characteristics of their binding (the binding constant, the Gibbs energy, enthalpy, entropy and heat capacity effect of binding) have also been determined. It is found that the binding of urea and guanidinium chloride by protein is accompanied by a significant decrease of enthalpy and entropy. At all concentrations of denaturants the enthalpy term slightly dominates the entropy term in the Gibbs energy function. Correlation analysis of the number of binding sites and structural characteristics of these proteins suggests that the binding sites for urea and guanidinium chloride are likely to be formed by several hydrogen bonding groups. This type of binding of the denaturant molecules should lead to a significant restriction of conformational freedom within the polypeptide chain. This raises a doubt as to whether a polypeptide chain in concentrated solutions of denaturants can be considered as a standard of a random coil conformation.     

pH corrections and protein ionization in water/guanidinium chloride.

More than 30 years ago, Nozaki and Tanford reported that the pK values for several amino acids and simple substances in 6 M guanidinium chloride differed little from the corresponding values in low salt (Nozaki, Y., and C. Tanford. 1967. J. Am. Chem. Soc. 89:736-742). This puzzling and counter-intuitive result hinders attempts to understand and predict the proton uptake/release behavior of proteins in guanidinium chloride solutions, behavior which may determine whether the DeltaG(N-D) values obtained from guanidinium chloride-induced denaturation data can actually be interpreted as the Gibbs energy difference between the native and denatured states (Bolen, D. W., and M. Yang. 2000. Biochemistry. 39:15208-15216). We show in this work that the Nozaki-Tanford result can be traced back to the fact that glass-electrode pH meter readings in water/guanidinium chloride do not equal true pH values. We determine the correction factors required to convert pH meter readings in water/guanidinium chloride into true pH values and show that, when these corrections are applied, the effect of guanidinium chloride on the pK values of simple substances is found to be significant and similar to that of NaCl. The results reported here allow us to propose plausible guanidinium chloride concentration dependencies for the pK values of carboxylic acids in proteins and, on their basis, to reproduce qualitatively the proton uptake/release behavior for the native and denatured states of several proteins (ribonuclease A, alpha-chymotrypsin, staphylococcal nuclease) in guanidinium chloride solutions. Finally, the implications of the pH correction for the experimental characterization of protein folding energetics are briefly discussed.     

Structural transitions of soluble and immobilized chymotrypsinogen A in urea and guanidinium chloride as measured by fluorescence

A cylindrical flow-through quartz cell was designed for measuring fluorescence changes associated with structural transitions in proteins immobilized by covalent attachment to insoluble matrices. Chymotrypsinogen A was immobilized by covalent attachment to derivatized porous glass beads. Conformational transitions in both native, soluble chymotrypsinogen and glass-bound chymotrypsinogen were assessed from fluorescence emission spectra obtained in 0 to 8 m urea and in 0 to 7 m guanidinium chloride. Evidence for the complete reversibility of such transitions in this zymogen was provided by comparing spectra generated by the native zymogen exposed to a given concentration of denaturant with spectra recorded for a mixture of the native zymogen and completely denatured zymogen at the same final concentration of denaturant. The observed transition appeared to follow a two-state mechanism. First order kinetics of unfolding and of refolding were observed in the transition region of the immobilized protein by monitoring fluorescence changes after rapidly adjusting the concentration of denaturant; apparent first order rate constants at pH 7 and 25 °C averaged 0.016 min^?1. Neither the chemistry of the immobilization reactions nor the microenvironment of the surface appears to affect the stability of the native zymogen or the refolding of denatured chymotrypsinogen. Thus, it appears that immobilization of proteins can provide a means for investigating conformational transitions which, due to such complicating secondary reactions as protein-protein interactions and autolysis, cannot otherwise be examined.     

Cation binding effects on the pH, thermal and urea denaturation transitions in alpha-lactalbumin

The binding of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations to bovine alpha-lactalbumin at 20 and 37 degrees C has been studied by means of intrinsic protein fluorescence. The values of apparent binding constants for these ions obtained at 37 degrees C are about one order of magnitude lower than those measured at 20 degrees C. Urea and alkali (pH greater than 10) induce unfolding transitions which involve stable partially unfolded intermediates for all metal ion-bound forms of alpha-lactalbumin. Heating induces similar partially unfolded states. Nevertheless, the partially unfolded states induced by heating, urea, alkaline or acidic treatments are somewhat different in their tryptophan residue environment properties. The results have been interpreted in terms of a simple scheme of equilibria between metal-free and metal-bound forms in their native, partially unfolded and unfolded states. The scheme provides an approach to the quantitative interpretation of any transition equilibrium shift induced by a low molecular mass species able to be bound by a protein.     

Comparison of inactivation and unfolding of green crab (Scylla serrata) alkaline phosphatase during denaturation by guanidinium chloride

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, each active site in which contains a tight cluster of two zinc ions and one magnesium ion. Unfolding and inactivation of the enzyme during denaturation in guanidinium chloride (GuHCl) solutions of different concentrations have been compared. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] has been applied to a study on the kinetics of the course of inactivation of the enzyme during denaturation by GuHCl. The rate constants of unfolding and inactivation have been determined. The results show that inactivation occurs before noticeable conformational change can be detected. It is suggested that the active site of green crab alkaline phosphatase containing multiple metal ions is also situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.