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.     

Coulomb forces control the density of the collapsed unfolded state of barstar

Although it has been recently shown that unfolded polypeptide chains undergo a collapse on transfer from denaturing to native conditions, the forces determining the dynamics and the size of the collapsed form have not yet been understood. Here, we use single-molecule fluorescence resonance energy transfer experiments on the small protein barstar to characterize the unfolded chain in guanidinium chloride (GdmCl) and urea. The unfolded protein collapses on decreasing the concentration of denaturants. Below the critical concentration of 3.5 M denaturant, the collapse in GdmCl leads to a more dense state than in urea. Since it is known that GdmCl suppresses electrostatic interactions, we infer that Coulomb forces are the dominant forces acting in the unfolded barstar under native conditions. This hypothesis is clearly buttressed by the finding of a compaction of the unfolded barstar by addition of KCl at low urea concentrations.     

Effect of denaturants on the 11S protein of guar seed (Cyamopsis tetragonoloba)

The effect of sodium dodecyl sulphate, urea or guanidinium hydrochloride on the sedimentation velocity, viscosity, ultra violet spectra and fluorescence spectra of the 11S protein of guar seed has been determined. Sodium dodecyl sulphate dissociates the protein directly to the 2S protein, whereas urea or guanidinium hydrochloride produces an intermediate 7S protein. These reagents denature the protein also. Both the dissociative and the denaturation effect follow the order, sodium dodecyl sulphate > guanidinium hydrochloride > urea when the concentration are expressed as mols per litre. The denatured states in the three cases probably differ.     

Cloning, Escherichia coli expression, purification, characterization, and enzyme assay of the ribosomal protein S4 from wheat seedlings (Triticum vulgare)

S4 is a paradigm of ribosomal proteins involved in multifarious activities both within and outside the ribosome. For a detailed biochemical and structural investigations of eukaryotic S4, the wheat S4 gene has been cloned and expressed in Escherichia coli, and the protein purified to a high degree of homogeneity. The 285-residue recombinant protein containing an N-terminal His(6) tag along with fourteen additional residues derived from the cloning vector is characterized by a molecular mass of 31981.24 Da. The actual sequence of 265 amino acids having a molecular mass of 29931 Da completely defines the primary structure of wheat S4. Homology modeling shows a bi-lobed protein topology arising from folding of the polypeptide into two domains, consistent with the fold topology of prokaryotic S4. The purified protein is stable and folded since it can be reversibly unfolded in guanidinium hydrochloride, and is capable of hydrolyzing cysteine protease-specific peptide-based fluorescence substrates, including Ac-DEVD-AFC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) and Z-FR-AMC (N-CBZ-Phe-Arg-aminomethylcoumarin).     

Reversible aggregation plays a crucial role on the folding landscape of p53 core domain

The role of tumor suppressor protein p53 in cell cycle control depends on its flexible and partially unstructured conformation, which makes it crucial to understand its folding landscape. Here we report an intermediate structure of the core domain of the tumor suppressor protein p53 (p53C) during equilibrium and kinetic folding/unfolding transitions induced by guanidinium chloride. This partially folded structure was undetectable when investigated by intrinsic fluorescence. Indeed, the fluorescence data showed a simple two-state transition. On the other hand, analysis of far ultraviolet circular dichroism in 1.0 M guanidinium chloride demonstrated a high content of secondary structure, and the use of an extrinsic fluorescent probe, 4,4'-dianilino-1,1' binaphthyl-5,5'-disulfonic acid, indicated an increase in exposure of the hydrophobic core at 1 M guanidinium chloride. This partially folded conformation of p53C was plagued by aggregation, as suggested by one-dimensional NMR and demonstrated by light-scattering and gel-filtration chromatography. Dissociation by high pressure of these aggregates reveals the reversibility of the process and that the aggregates have water-excluded cavities. Kinetic measurements show that the intermediate formed in a parallel reaction between unfolded and folded structures and that it is under fine energetic control. They are not only crucial to the folding pathway of p53C but may explain as well the vulnerability of p53C to undergo departure of the native to an inactive state, which makes the cell susceptible to malignant transformation.     

The effect of denaturants on protein structure

Virtually all studies of the protein-folding reaction add either heat, acid, or a chemical denaturant to an aqueous protein solution in order to perturb the protein structure. When chemical denaturants are used, very high concentrations are usually necessary to observe any change in protein structure. In a solution with such high denaturant concentrations, both the structure of the protein and the structure of the solvent around the protein can be altered. X-ray crystallography is the obvious experimental technique to probe both types of changes. In this paper, we report the crystal structures of dihydrofolate reductase with urea and of ribonuclease A with guanidinium chloride. These two classic denaturants have similar effects on the native structure of the protein. The most important change that occurs is a reduction in the overall thermal factor. These structures offer a molecular explanation for the reduction in mobility. Although the reduction is observed only with the native enzyme in the crystal, a similar decrease in mobility has also been observed in the unfolded state in solution (Makhatadze G, Privalov PL. 1992. Protein interactions with urea and guanidinium chloride: A calorimetric study. J Mol Biol 226:491-505).     

Lipid requirements for the structure and function of the fatty acid synthetase complex from Ceratitis capitata.

Lipid content of purified fatty acid synthetase preparations from the Dipterous Ceratitis capitata correlated with the enzyme activity. Delipidation of the enzyme by extracting with a series of organic solvents rendered a protein without any residual activity and the treatment with phospholipase A2 for 30 min reduced the activity to 10%. Addition of lipid classes to either the native enzyme or the phospholipase-treated preparation enhanced the activity in a different manner, phosphatidylethanolamine being the most effective lipid. The role of the lipids in the lipoprotein structure of the complex was studied by circular dichroism spectra of the native enzyme and in the presence of a concentration range of urea, guanidinium chloride, sodium dodecyl sulfate, sodium cholate, and sodium chloride. 3 M urea and 1.5 M guanidinium chloride induced a conformational transition of the lipoprotein that lost its alpha-helical structure at higher concentrations of both reagents. Sodium dodecyl sulfate and sodium cholate had little effect on the alpha-helical structure, although both reagents induced the loss of enzyme activity. Cholate had essentially the same effect as phospholipids on the maintenance of the native structure but it was unable to support the enzyme activity.     

Inclusion bodies of the thermophilic endoglucanase D from Clostridium thermocellum are made of native enzyme that resists 8 M urea.

Endoglucanase D from Clostridium thermocellum was purified from inclusion bodies formed upon its overproduction in Escherichia coli, using 5 M urea as a solubilizing solution. We examined the effects of denaturing agents upon the stability of the pure soluble enzyme as a function of the temperature. At room temperature, guanidinium chloride induces an irreversible denaturation. By comparison, we observed no structural or functional effects at room temperature using high concentrations of urea as denaturing agent. The irreversible denaturation process observed with guanidinium chloride also occurs with urea but only at elevated temperature (greater than or equal to 60 degrees C); in 6 M urea, the activation energy of the denaturation reaction is decreased by a factor of only 1.8. We interpret the high resistance of this protein to urea as reflecting a reduced flexibility of its structure at normal temperatures which should be correlated to the thermophilic origin of this protein.     

Conformational changes in intact and papain-modified alpha 1-proteinase inhibitor induced by guanidinium chloride

Equilibrium unfolding-refolding processes of active and proteolytically modified alpha 1-proteinase inhibitor induced by guanidinium chloride were studied. Spectroscopic methods of ultraviolet absorption, fluorescence emission and circular dichroism were used. The functional inhibitor unfolds following a multistate process: a first transition (midpoint at 0.6 M guanidinium chloride) was observed whatever the method used and was attributed to a limited conformational modification of the region including the two tryptophan residues. At higher denaturant concentrations, two other transitions were observed, one in fluorescence (midpoint at 1.7 M guanidinium chloride), attributed to the unfolding of the polypeptide chain in the same region and the other one, observed in circular dichroism and in ultraviolet absorption (midpoint at 2.3 M guanidinium chloride), leading to the totally unfolded protein. Evidence for several intermediates was also obtained with the proteolytically modified inhibitor. If total unfolding is considered, the modified inhibitor was found to be more stable towards the denaturant than the functional form (obtained at 5.5 M and 3.5 M guanidinium chloride, respectively). The unfolding irreversibility observed was attributed to the C-terminal fragment Ser359-Lys394 associated with the main chain of the cleaved inhibitor.     

Guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase: stabilization of an apo-protein by GdmCl

The guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase, was examined in detail using multiple spectroscopic techniques, enzyme activity measurements and size exclusion chromatography. The equilibrium unfolding of FprA by urea is a cooperative process where no stabilization of any partially folded intermediate of protein is observed. In comparison, the unfolding of FprA by guanidinium chloride proceeds through intermediates that are stabilized by interaction of protein with guanidinium chloride. In the presence of low concentrations of guanidinium chloride the protein undergoes compaction of the native conformation; this is due to optimization of charge in the native protein caused by electrostatic shielding by the guanidinium cation of charges on the polar groups located on the protein side chains. At a guanidinium chloride concentration of about 0.8 m, stabilization of apo-protein was observed. The stabilization of apo-FprA by guanidinium chloride is probably the result of direct binding of the Gdm+ cation to protein. The results presented here suggest that the difference between the urea- and guanidinium chloride-induced unfolding of FprA could be due to electrostatic interactions stabilizating the native conformation of this protein.     

Effect of denaturants on the oligomeric structure of the 11 S protein of sunflower (Helianthus annum)

The effect of sodium dodecyl sulphate, urea, guanidinium hydrochloride and heat on the oligomeric structure of the 11 S protein of sunflower has been determined. Sodium dodecyl sulphate directly dissociates the protein to 2 S subunits, whereas urea and guanidinium hydrochloride dissociate it through an intermediate 7 S protein. Heating the protein at 90‡C for 20 min caused dissociation of the 11 S protein, without any precipitation.     

Guanidinium chloride and urea denaturations of beta-lactoglobulin A at pH 2.0 and 25 degrees C: the equilibrium intermediate contains non-native structures (helix, tryptophan and hydrophobic patches)

We have carried out guanidinium chloride (GdmCl) and urea denaturations of bovine beta-lactoglobulin A (beta-lgA) at pH 2.0 and 25 degrees C, using far-UV and near-UV circular dichroism, near-UV absorption and tryptophan fluorescence spectroscopies. The stable intermediate state that occurs during GdmCl denaturation has been characterized by the far- and near-UV circular dichroism, tryptophan difference absorption, tryptophan fluorescence and 8-anilino-1-naphthalene sulphonic acid binding measurements. Following conclusions have been reached. (a) Urea-induced denaturation is not a two-state process. (b) GdmCl-induced denaturation is composed of two distinct two-state processes. (c) alpha-Helical content, burial of tryptophan residues and burial of hydrophobic surface area are more in the GdmCl-induced stable intermediate than those originally present in the native protein.     

Molecular basis for the effect of urea and guanidinium chloride on the dynamics of unfolded polypeptide chains

Chemical denaturants are frequently used to unfold proteins and to characterize mechanisms and transition states of protein folding reactions. The molecular basis of the effect of urea and guanidinium chloride (GdmCl) on polypeptide chains is still not well understood. Models for denaturant--protein interaction include both direct binding and indirect changes in solvent properties. Here we report studies on the effect of urea and GdmCl on the rate constants (k(c)) of end-to-end diffusion in unstructured poly(glycine-serine) chains of different length. Urea and GdmCl both lead to a linear decrease of lnk(c) with denaturant concentration, as observed for the rate constants for protein folding. This suggests that the effect of denaturants on chain dynamics significantly contributes to the denaturant-dependence of folding rate constants for small proteins. We show that this linear dependency is the result of two additive non-linear effects, namely increased solvent viscosity and denaturant binding. The contribution from denaturant binding can be quantitatively described by Schellman's weak binding model with binding constants (K) of 0.62(+/-0.01)M(-1) for GdmCl and 0.26(+/-0.01)M(-1) for urea. In our model peptides the number of binding sites and the effect of a bound denaturant molecule on chain dynamics is identical for urea and GdmCl. The results further identify the polypeptide backbone as the major denaturant binding site and give an upper limit of a few nanoseconds for residence times of denaturant molecules on the polypeptide chain.     

Molecular basis for the polymerization of octopus lens S-crystallin

S-Crystallin from octopus lens has a tertiary structure similar to sigma-class glutathione transferase (GST). However, after isolation from the lenses, S-crystallin was found to aggregate more easily than sigma-GST. In vitro experiments showed that the lens S-crystallin can be polymerized and finally denatured at increasing concentration of urea or guanidinium chloride (GdmCl). In the intermediate concentrations of urea or GdmCl, the polymerized form of S-crystallin is aggregated, as manifested by the increase in light scattering and precipitation of the protein. There is a delay time for the initiation of polymerization. Both the delay time and rate of polymerization depend on the protein concentration. The native protein showed a maximum fluorescence emission spectrum at 341 nm. The GdmCl-denatured protein exhibited two fluorescence maxima at 310 nm and 358 nm, respectively, whereas the urea-denatured protein showed a fluorescence peak at 358 nm with a small peak at 310 nm. The fluorescence intensity was quenched. Monomers, dimers, trimers, and polymers of the native protein were observed by negative-stain electron microscopic analysis. The aggregated form, however, showed irregular structure. The aggregate was solubilized in high concentrations of urea or GdmCl. The redissolved denatured protein showed an identical fluorescence spectrum to the protein solution that was directly denatured with high concentrations of urea or GdmCl. The denatured protein was readily refolded to its native state by diluting with buffer solution. The fluorescence spectrum of the renatured protein solution was similar to that of the native form. The phase diagrams for the S-crystallin in urea and GdmCl were constructed. Both salt concentration and pH value of the solution affect the polymerization rate, suggesting the participation of ionic interactions in the polymerization. Comparison of the molecular models of the S-crystallin and sigma-GST suggests that an extra ion-pair between Asp-101 and Arg-14 in S-crystallin contributes to stabilizing the protomer. Furthermore, the molecular surface of S-crystallin has a protruding Lys-208 on one side and a complementary patch of aspartate residues (Asp-90, Asp-94, Asp-101, Asp-102, Asp-179, and Asp-180) on the other side. We propose a molecular model for the S-crystallin polymer in vivo, which involves side-by-side associations of Lys-208 from one protomer and the aspartate patch from another protomer that allows the formation of a polymeric structure spontaneously into a liquid crystal structure in the lens.     

Allosteric serine hydroxymethyltransferase from monkey liver: Correlation of conformational changes caused by denaturants with the alterations in catalytic activity

The far-ultraviolet region circular dichroic spectrumof serine hydroxymethyltransferase from monkey liver showed that the protein is in an α-helical conformation. The near ultraviolet circular dichoric spectrum revealed two negative bands originating from the tertiary conformational environment of the aromatic amino acid residues. Addition of urea or guanidinium chloride perturbed the characteristic fluorescence and far ultraviolet circular dichroic spectrum of the enzyme. The decrease in (θ)₂₂₂ and enzyme activity followed identical patterns with increasing concentrations of urea, whereas with guanidinium chloride, the loss of enzyme activity preceded the loss of secondary structure. 2-Chloroethanol, trifluoroethanol and sodium dodecyl sulphate enhanced the mean residue ellipticity values. In addition, sodium dodecyl sulphate also caused a perturbation of the fluorescence emission spectrum of the enzyme. Extremes of pH decreased the — (θ)₂₂₂ value. Plots of — (θ)₂₂₂and enzyme activity as a function of pH showed maximal values at pH 7.4–7.5. These results suggested the prevalence of “conformational flexibility” in the structure of serine hydroxymethyltransferase.     

TEM-1 beta-lactamase folds in a nonhierarchical manner with transient non-native interactions involving the C-terminal region

The conformational stability and kinetics of refolding and unfolding of the W290F mutant of TEM-1 beta-lactamase have been determined as a function of guanidinium chloride concentration. The activity and spectroscopic properties of the mutant enzyme did not differ significantly from those of the wild type, indicating that the mutation has only a very limited effect on the structure of the protein. The stability of the folded protein is reduced, however, by 5-10 kJ mol-1 relative to that of the molten globule intermediate (H), but the values of the folding rate constants are unchanged, suggesting that Trp-290 becomes organized in its nativelike environment only after the rate-limiting step; i.e., the C-terminal region of the enzyme folds very late. In contrast to the significant increase in fluorescence intensity seen in the dead time (3-4 ms) of refolding of the wild-type protein, no corresponding burst phase was observed with the mutant enzyme, enabling the burst phase to be attributed specifically to the C-terminal Trp-290. This residue is suggested to be buried in a nonpolar environment from which it has to escape during subsequent folding steps. With both proteins, fast early collapse leads to a folding intermediate in which the C-terminal region of the polypeptide chain is trapped in a non-native structure, consistent with a nonhierarchical folding process.     

Multiple unfolding states of glutathione transferase from Physa acuta (Gastropoda [correction of Gastropada]: Physidae)

The equilibrium unfolding of the major Physa acuta glutathione transferase isoenzyme (P. acuta GST(3)) has been performed using guanidinium chloride (GdmCl), urea, and acid denaturation to investigate the unfolding intermediates. Protein transitions were monitored by intrinsic fluorescence. The results indicate that unfolding of P. acuta GST(3) using GdmCl (0-3.0M) is a multistep process, i.e., three intermediates coexist in equilibrium. The first intermediate, a partially dissociated dimer, exists at low GdmCl concentration (approximately at 0.7M). At 1.2M GdmCl, a dimeric intermediate with a compact structure was observed. This intermediate undergoes dissociation into structural monomers at 1.75M of GdmCl. The monomeric intermediate started to be completely unfolding at higher GdmCl concentrations (>1.8M). Unfolding using urea (0-7.0M) and acid-induced structures as well as the fluorescence of 8-anilino-1-naphthalenesulfonate in the presence of different GdmCl concentrations confirmed that the unfolding is a multistep process. At concentrations of GdmCl or urea less than the midpoints or at the midpoint pH (pH 4.2-4.6), the unfolding transition is protein concentration independent and involved a change in the subunit tertiary structure yielding a partially active dimeric intermediate. The binding of glutathione to the enzyme active site stabilizes the native dimeric state.     

Dissecting contributions to the denaturant sensitivities of proteins

Understanding the molecular basis for protein denaturation by urea and guanidinium chloride (GdmCl) should accommodate the observation that, on a molar basis, GdmCl is generally 2-2.5-fold more effective as a protein denaturant than urea. Previous studies [Smith, J. S., and Scholtz, J. M. (1996) Biochemistry 35, 7292-7297] have suggested that the effects of GdmCl on the stability of alanine-based helical peptides can be separated into denaturant and salt effects, since adding equimolar NaCl to urea enhanced urea-induced unfolding to an extent that was close to that of Gdm. We reinvestigated this observation using an alanine-based helical peptide (alahel) that lacks side chain electrostatic contributions to stability, and compared the relative denaturant sensitivities of this peptide with that of tryptophan zipper peptides (trpzip) whose native conformations are stabilized largely by cross-strand indole ring interactions. In contrast to the observations of Smith and Scholtz, GdmCl was only slightly more powerful as a denaturant of alahel than urea in salt-free buffer (the denaturant m value m(GdmCl)/m(urea) ratio = 1.4), and the denaturation of alahel by urea exhibited only a small dependence on NaCl or KCl. The trpzip peptides were much more sensitive to GdmCl than to urea (m(GdmCl)/m(urea) = 3.5-4). These observations indicate that the m(GdmCl)/m(urea) ratio of 2-2.5 for proteins results from a combination of effects on the multiple contributions to protein stability, for which GdmCl may be only slightly more effective than urea (e.g., hydrogen bonds) or considerably more effective than urea (e.g., indole-indole interactions).     

Denaturation and renaturation of the monomeric phosphoglycerate mutase from Schizosaccharomyces pombe.

The denaturation by guanidinium chloride of the monomeric phosphoglycerate mutase from Schizosaccharomyces pombe was studied. The loss in activity broadly parallels the changes in protein structure detected by fluorescence and c.d. Renaturation can be brought about by dilution of the denaturing agent. These processes were compared with those in the enzymes from baker's yeast and rabbit muscle, which are tetrameric and dimeric respectively. The effects of the cofactor 2,3-bisphosphoglycerate on the structure and stability of the S. pombe enzyme were also investigated.     

Calorimetric study of the heat and cold denaturation of beta-lactoglobulin.

Temperature-induced changes of the states of beta-lactoglobulin have been studied calorimetrically. In the presence of a high concentration of urea this protein shows not only heat but also cold denaturation. Its heat denaturation is approximated very closely by a two-state transition, while the cold denaturation deviates considerably from the two-state transition and this deviation increases as the temperature decreases. The heat effect of cold denaturation is opposite in sign to that of heat denaturation and is noticeably larger in magnitude. This difference in magnitude is caused by the temperature-dependent negative heat effect of additional binding of urea to the polypeptide chain of the protein upon its unfolding, which decreases the positive enthalpy of heat denaturation and increases the negative enthalpy of cold denaturation. The binding of urea considerably increases the partial heat capacity of the protein, especially in the denatured state. However, when corrected for the heat capacity effect of urea binding, the partial heat capacity of the denatured protein is close in magnitude to that expected for the unfolded polypeptide chain in aqueous solution without urea but only for temperatures below 10 degrees C. At higher temperatures, the heat capacity of the denatured protein is lower than that expected for the unfolded polypeptide chain. It appears that at temperatures above 10 degrees C not all the surface of the beta-lactoglobulin polypeptide chain is exposed to the solvent, even in the presence of 6 M urea; i.e., the denatured protein is not completely unfolded and unfolds only at temperatures lower than 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)