Does beta-lactoglobulin denaturation occur via an intermediate state?

The denaturation of beta-lactoglobulin (BLG) in the presence of urea and GuHCl has been investigated at different pH values with various spectroscopic techniques. The equilibrium denaturation free energy values, obtained by linearly extrapolating the data to vanishing denaturant (DeltaG(D)(H2O)), are compared and discussed. The fit of the spectroscopic data monitoring the denaturation of BLG has been approached, at first, with a two-state model that describes the protein transition from the folded state (at each pH and in the absence of denaturant) to the denatured state, but in particular, along the GuHCl denaturation pathway some evidence is found of the presence of an intermediate state. Time-resolved fluorescence experiments performed on the BLG-ANS (1-anilino-8-naphthalenesulfonate) complex help to understand the results. Fluorescence polarization anisotropy (FPA) measurements accompanying the denaturation process show the presence of a fast rotational diffusion of the ANS probe, and the data are interpreted in terms of local fluctuations of a still structured tract of the denatured protein where the probe is bound.     

Thermal stabilities of globular proteins

Statistical thermodynamic theory has recently been developed to account for the stabilities of globular proteins. Here we extend that work to predict the dependence on temperature. Folding is assumed to be driven by solvophobic interactions and opposed by the conformational entropy. The temperature dependence of the solvophobic interaction is taken from the transfer experiments on amino acids by Tanford and Nozaki and on model solutes by Gill and Wads?. One long-standing puzzle has been why proteins denature upon heating, since the solvophobic force to fold strengthens with increasing temperature. This is resolved by the theory, which predicts two first-order phase transitions. "Cold denaturation" is driven principally by the weakening of the solvophobic interaction, but normal denaturation is driven principally by the gain of conformational entropy of the chain. Predictions of the thermodynamic state functions are in reasonable agreement with the calorimetric experiments of Privalov and Khechinashvili. Comparison of the theory with experiments suggests that there may be an additional enthalpic driving force toward folding which is not due to the solvophobic interactions.     

Reshaping the folding energy landscape by chloride salt: impact on molten-globule formation and aggregation behavior of carbonic anhydrase

During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used. Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl. On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea). However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl. Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa). Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt. Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state. Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase. Refolding from this state only provided low recoveries of native enzyme.     

Estimation of the stability of globular proteins

The interpretation of ΔG (the free energy change for the reaction, globular conformation ? randomly coiled conformation, in the absence of denaturant), in terms of the free energies of transfer of various parts of the protein molecule from water to denaturant solution, is unsatisfactory because the latter are assumed to be identical to the transfer-free energies of similar groups attached to smaller model compounds. We have made empirical adjustments to transfer-free energy theory that make possible linear extrapolation of the free energy of denaturation of a protein from transition region to zero denaturant concentration. The modified theory, used to analyze the denaturation of proteins by guanidine hydrochloride and urea, allowed us to calculate reasonable values for Δα, the average change in accessibility to solvent of the component groups of protein.     

Effects of denaturants at low concentrations on the reversible denaturation of staphylococcal nuclease

Three very unstable mutant forms of staphylococcal nuclease were used to quantitate the change in the apparent equilibrium constant for reversible denaturation (Kapp) as a function of denaturant concentration for a variety of different denaturing solutes. The value of this equilibrium constant in the absence of denaturant (Kapp,0) was determined by renaturation of the mutant proteins with a combination of glycerol and calcium ion, the latter of which binds at the active site in the native conformation. Because Kapp,0 fell in the easily measurable range between 0.1 and 1, the change in Kapp, and thus the change in free energy (delta Gapp), at very low concentrations of denaturants could be accurately measured. With guanidine hydrochloride (GuHCl), the rate of change of the apparent free energy of denaturation with respect to denaturant concentration (d(delta Gapp)/dCGuHCl or mGuHCl) was found to be remarkably constant down to zero denaturant concentration, even though this value was different for each of the three proteins. Unlike GuHCl, urea exhibited a slightly reduced value of d delta Gapp/dCurea at low concentrations. Results with a number of thiocyanate, perchlorate, and iodide salts confirmed that the Hofmeister series holds for concentrations below 0.1 M; that is, with regard to efficacy as a denaturant SCN- greater than ClO4- greater than I- and Li+,NH4+ greater than Na+,K+. However, all of the chaotropic salts analyzed exhibited markedly increased values of d(delta Gapp)/dCsalt at concentrations below 0.2 M. One possible explanation for these large deviations from a linear relationship between delta Gapp and salt concentration is that weak binding or adsorption of chaotropic anions is occurring at a saturable number of sites in hydrophobic regions of the denatured state.     

Theory for the folding and stability of globular proteins

Using lattice statistical mechanics, we develop theory to account for the folding of a heteropolymer molecule such as a protein to the globular and soluble state. Folding is assumed to be driven by the association of solvophobic monomers to avoid solvent and opposed by the chain configurational entropy. Theory predicts a phase transition as a function of temperature or solvent character. Molecules that are too short or too long or that have too few solvophobic residues are predicted not to fold. Globular molecules should have a largely solvophobic core, but there is an entropic tendency for some residues to be "out of place", particularly in small molecules. For long chains, molecules comprised of globular domains are predicted to be thermodynamically more stable than spherical molecules. The number of accessible conformations in the globular state is calculated to be an exceedingly small fraction of the number available to the random coil. Previous estimates of this number, which have motivated kinetic theories of folding, err by many tens of orders of magnitude.     

Apparent pKa shifts of titratable residues at high denaturant concentration and the impact on protein stability

Urea and guanidine-hydrochloride (GdnHCl) are frequently used for protein denaturation in order to determine the Gibbs free energy of folding and kinetic folding/unfolding parameters. Constant pH value is applied in the folding/unfolding experiments at different denaturant concentrations and steady protonation state of titratable groups is assumed in the folded and unfolded protein, respectively. The apparent side-chain pKa values of Asp, Glu, His and Lys in the absence and presence of 6 M urea and GdnHCl, respectively, have been determined by 1H-NMR. pKa values of all four residues are up-shifted by 0.3-0.5 pH units in presence of 6 M urea by comparison with pKa values of the residues dissolved in water. In the presence of 6 M GdnHCl, pKa values are down-shifted by 0.2-0.3 pH units in the case of acidic and up-shifted by 0.3-0.5 pH units in the case of basic residues. Shifted pKa values in the presence of denaturant may have a pronounced effect on the outcome of the protein stability obtained from denaturant unfolding experiments.     

Reversible denaturation of oligomeric human chaperonin 10: denatured state depends on chemical denaturant

Chaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides. Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure. We have characterized denaturation and renaturation of the recombinant human chaperonin 10 (cpn10), which forms a heptamer. Denaturation induced by chemical denaturants urea and guanidine hydrochloride (GuHCl) as well as by heat was monitored by tyrosine fluorescence, far-ultraviolet circular dichroism, and cross-linking; all denaturation reactions were reversible. GuHCl-induced denaturation was found to be cpn10 concentration dependent, in accord with a native heptamer to denatured monomer transition. In contrast, urea-induced denaturation was not cpn10 concentration dependent, suggesting that under these conditions cpn10 heptamers denature without dissociation. There were no indications of equilibrium intermediates, such as folded monomers, in either denaturant. The different cpn10 denatured states observed in high [GuHCl] and high [urea] were supported by cross-linking experiments. Thermal denaturation revealed that monomer and heptamer reactions display the same enthalpy change (per monomer), whereas the entropy-increase is significantly larger for the heptamer. A thermodynamic cycle for oligomeric cpn10, combining chemical denaturation with the dissociation constant in absence of denaturant, shows that dissociated monomers are only marginally stable (3 kJ/mol). The thermodynamics for co-chaperonin stability appears conserved; therefore, instability of the monomer could be necessary to specify the native heptameric structure.     

Thermodynamics of denaturation of α-chymotrypsinogen A in aqueous urea and alkylurea solutions

The effects of pH, urea, and alkylureas on the thermal stability ofα-chymotrypsinogen A (α-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition ofα-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25°C were calculated. Comparison of these values shows that at 25°C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein molecules. At pH lower than 2.0,α-ctg A still exists in a very stable form, probably the acid-denatured form (A-form).     

Investigation of the four cooperative unfolding units existing in the MICAL-1 CH domain

The solution structure of human MICAL-1 calpolnin homology (CH) domain is composed of six alpha helices and one 3(10) helix. To study the unfolding of this domain, we carry out native-state hydrogen exchange, intrinsic fluorescence and far-UV circular dichroism experiments. The free energy of unfolding, DeltaG(H2O), is calculated to be 7.11+/-0.58 kcal mol(-1) from GuHCl denaturation at pH 6.5. Four cooperative unfolding units are found using native-state hydrogen exchange experiment. Forty-seven slow-exchange residues can be studied by native-state hydrogen exchange experiments. From the concentration dependence of exchange rates, free energy of amide hydrogen with solvent, DeltaG(HX) and m-value (sensitivity of exposure to denaturant) are obtained, which reveal four cooperative unfolding units. The slowest exchanging protons are distributed throughout the whole hydrophobic core of the protein, which might be the folding core. These results will help us understand the structure of MICAL-1 CH domain more deeply.     

Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation.

The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, delta Gu (H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing delta Gu (H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed.     

Thermodynamic strategies for stabilizing intermediate states of proteins

This paper presents three theorems pertaining to thermodynamic properties of the intermediate (e.g., molten globule) state of proteins exhibiting such a conformation in the presence of GuHCl or urea. The theorems are proved for the three-state case using the denaturant binding model and the linear extrapolation model; their utility is illustrated via applications to examples in the literature. Theorem One states that the denaturant activity that maximizes the population of a partly folded conformation is at any temperature independent of the Gibbs free energy difference between the intermediate and native states. This result holds for both models of protein–denaturant interaction. The second theorem claims that the population maximum is independent, of the denaturant association constant for the denaturant binding model. Theorem Three, which also applies to both models considered here, states that at the temperatures corresponding to the extrema in the population of the intermediate, the enthalpy change of the intermediate is equal to the excess enthalpy function, an experimentally accessible quantity. In the absence of denaturant, the enthalpy change of the intermediate state at the population extrema can be written as a function of the thermodynamic parameters of the unfolded state alone. These results, which can be applied to systems of any number of states under certain conditions, should aid in the optimization of conditions employed for experimental studies of partly organized states of proteins. © 1994 John Wiley & Sons, Inc.     

NMR studies on the induced denaturation of lysozyme by guanidine hydrochloride in the presence of the inhibitor (NAG)3

The stabilization of the folded conformation of lysozyme, arising from the binding of the inhibitor (NAG)3 against induced denaturation, is demonstrated from the 1H-nmr spectra of the enzyme. The nmr spectra reveal that the binding of the denaturant (GuHCl) to the enzyme is associated with changes in the conformation of the enzyme. The binding site of the inhibitor site C also serves as one of the binding sites of GuHCl. The observation that higher denaturant concentrations are required in the unfolding of Lys-(NAG)3 as compared to free Lys can be explained partly in terms of the existence of a competitive binding to the enzyme involving the (NAG)3 and GuHCl molecules.     

Intrinsically disordered structure of Bacillus pasteurii UreG as revealed by steady-state and time-resolved fluorescence spectroscopy

UreG is an essential protein for the in vivo activation of urease. In a previous study, UreG from Bacillus pasteurii was shown to behave as an intrinsically unstructured dimeric protein. Here, intrinsic and extrinsic fluorescence experiments were performed, in the absence and presence of denaturant, to provide information about the form (fully folded, molten globule, premolten globule, or random coil) that the native state of BpUreG assumes in solution. The features of the emission band of the unique tryptophan residue (W192) located on the C-terminal helix, as well as the rate of bimolecular quenching by potassium iodide, indicated that, in the native state, W192 is protected from the aqueous polar solvent, while upon addition of denaturant, a conformational change occurs that causes solvent exposure of the indole side chain. This structural change, mainly affecting the C-terminal helix, is associated with the release of static quenching, as shown by resolution of the decay-associated spectra. The exposure of protein hydrophobic sites, monitored using the fluorescent probe bis-ANS, indicated that the native dimeric state of BpUreG is disordered even though it maintains a significant amount of tertiary structure. ANS fluorescence also indicated that, upon addition of a small amount of GuHCl, a transition to a molten globule state occurs, followed by formation of a pre-molten globule state at a higher denaturant concentration. The latter form is resistant to full unfolding, as also revealed by far-UV circular dichroism spectroscopy. The hydrodynamic parameters obtained by time-resolved fluorescence anisotropy at maximal denaturant concentrations (3 M GuHCl) confirmed the existence of a disordered but stable dimeric protein core. The nature of the forces holding together the two monomers of BpUreG was investigated. Determination of free thiols in native or denaturant conditions, as well as light scattering experiments in the absence and presence of dithiothreitol as a reducing agent, under native or denaturing conditions, indicates that a disulfide bond, involving the unique conserved cysteine C68, is present under native conditions and maintained upon addition of denaturant. This covalent bond is therefore important for the stabilization of the dimer under native conditions. The intrinsically disordered structure of UreG is discussed with respect to the role of this protein as a chaperone in the urease assembly system.     

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.     

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).     

Small-angle X-ray scattering and single-molecule FRET spectroscopy produce highly divergent views of the low-denaturant unfolded state

The results of more than a dozen single-molecule F?rster resonance energy transfer (smFRET) experiments suggest that chemically unfolded polypeptides invariably collapse from an expanded random coil to more compact dimensions as the denaturant concentration is reduced. In sharp contrast, small-angle X-ray scattering (SAXS) studies suggest that, at least for single-domain proteins at non-zero denaturant concentrations, such compaction may be rare. Here, we explore this discrepancy by studying protein L, a protein previously studied by SAXS (at 5?°C), which suggested fixed unfolded-state dimensions from 1.4 to 5?M guanidine hydrochloride (GuHCl), and by smFRET (at 25?°C), which suggested that, in contrast, the chain contracts by 15-30% over this same denaturant range. Repeating the earlier SAXS study under the same conditions employed in the smFRET studies, we observe little, if any, evidence that the unfolded state of protein L contracts as the concentration of GuHCl is reduced. For example, scattering profiles (and thus the shape and dimensions) collected within ～4?ms after dilution to as low as 0.67?M GuHCl are effectively indistinguishable from those observed at equilibrium at higher denaturant. Our results thus argue that the disagreement between SAXS and smFRET is statistically significant and that the experimental evidence in favor of obligate polypeptide collapse at low denaturant cannot be considered conclusive yet.     

Polar or Apolar—The Role of Polarity for Urea-Induced Protein Denaturation

Urea-induced protein denaturation is widely used to study protein folding and stability; however, the molecular mechanism and driving forces of this process are not yet fully understood. In particular, it is unclear whether either hydrophobic or polar interactions between urea molecules and residues at the protein surface drive denaturation. To address this question, here, many molecular dynamics simulations totalling ca. 7 µs of the CI2 protein in aqueous solution served to perform a computational thought experiment, in which we varied the polarity of urea. For apolar driving forces, hypopolar urea should show increased denaturation power; for polar driving forces, hyperpolar urea should be the stronger denaturant. Indeed, protein unfolding was observed in all simulations with decreased urea polarity. Hyperpolar urea, in contrast, turned out to stabilize the native state. Moreover, the differential interaction preferences between urea and the 20 amino acids turned out to be enhanced for hypopolar urea and suppressed (or even inverted) for hyperpolar urea. These results strongly suggest that apolar urea–protein interactions, and not polar interactions, are the dominant driving force for denaturation. Further, the observed interactions provide a detailed picture of the underlying molecular driving forces. Our simulations finally allowed characterization of CI2 unfolding pathways. Unfolding proceeds sequentially with alternating loss of secondary or tertiary structure. After the transition state, unfolding pathways show large structural heterogeneity.     

Temperature- and denaturant-induced unfolding of two thermophilic esterases.

We studied the temperature- and denaturant-induced denaturation of two thermophilic esterases, AFEST from Archeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, by means of circular dichroism measurements. Both enzymes showed a very high denaturation temperature: 99 degrees C for AFEST and 91 degrees C for EST2. They also showed a remarkable resistance against urea; at half-completion of the transition the urea concentration was 7.1 M for AFEST and 5.9 M for EST2. On the contrary, both enzymes showed a weak resistance against GuHCl; at half-completion of the transition the GuHCl concentration was 2.0 M for AFEST and 1.9 M for EST2. The thermodynamic parameters characterizing urea- and GuHCl-induced denaturation of the studied enzymes have been obtained by both the linear extrapolation model and the denaturant binding model. The dependence of the thermal stability on NaCl concentration for both esterases has also been determined. A careful analysis of the data, coupled with available structural information, has allowed the proposal of a reliable interpretation.