Denaturant Binding Model Research Articles

A Structure-Based Mechanism for the Denaturing Action of Urea, Guanidinium Ion and Thiocyanate Ion.

An exhaustive analysis of all the protein structures deposited in the Protein Data Bank, here performed, has allowed the identification of hundredths of protein-bound urea molecules and the structural characterization of such binding sites. It emerged that, even though urea molecules are largely involved in hydrogen bonds with both backbone and side chains, they are also able to make van der Waals contacts with nonpolar moieties. As similar findings have also been previously reported for guanidinium and thiocyanate, this observation suggests that promiscuity is a general property of protein denaturants. Present data provide strong support for a mechanism based on the protein-denaturant direct interactions with a denaturant binding model to equal and independent sites. In this general framework, our investigations also highlight some interesting insights into the different denaturing power of urea compared to guanidinium/thiocyanate.

Free energy landscape of lysozyme: multiple near-native conformational states and rollover in the urea dependence of folding energy.

Deviation from linearity of the equilibrium folding free energy (ΔG) of proteins along the reaction coordinate is scarcely known. Optical spectroscopic observables and NMR-measured average molecular dimensional property of lysozyme with urea at pH 5 reveal that ΔG rolls over from linearity under mild to strongly native-like conditions. The urea dependence of ΔG is graphed in the 0-7 M range of the denaturant by employing a series of guanidine hydrochloride (GdnHCl)-induced equilibrium unfolding transitions, each in the presence of a fixed level of urea. The observed linear dependence of ΔG on urea under denaturing conditions begins to deviate as moderately native-like conditions are approached and eventually rolls over under strongly native-like conditions. This is atypical of the upward curvature in the ΔG vs denaturant plot predicted by the denaturant binding model. On increasing the denaturant concentration from 0 to 5 M, the hydrodynamic radius of lysozyme shrinks by ∼2 Å. We suggest subdenaturing levels of urea affect the population distribution among multiple near-native isoenergetic conformational states so as to promote them sequentially with increments of the denaturant. We use a multiple-state sequential model to show that the keel over of ΔG occurs due to these near-native alternative states in the native ensemble used for defining the unfolding equilibrium constant (KU), which we assume to vary linearly with urea. The results and the model appear to indicate a rugged flat bottom in the free energy landscape wherein population distribution of native-like states is modulated by urea-affected interstate motions.

Entropic stabilization of myoglobin by subdenaturing concentrations of guanidine hydrochloride

To find out the changes in the internal dynamics and function of proteins as a consequence of their binding interactions with guanidine hydrochloride (GdnHCl), laser flash photolysis and optical absorption methods have been used to study the dynamic events in the horse myoglobin-CO complex (MbCO) in the presence of subdenaturing concentrations of GdnHCl at pH 7, 22 degrees C. The rate coefficients for geminate rebinding and bimolecular rebinding (k(on)) were measured by laser photolysis of CO in MbCO, and the CO dissociation rate (k(off)) was determined by the CO replacement method using hexacyanoferrate ion or NO. Starting from the native-state condition, the values of k(on) and k(off) decrease by approximately 1.4 (+/-0.1)-fold in the presence of 0.1-0.3 M GdnHCl, and then increase at higher concentrations of the denaturant. This has been taken as evidence for internal motional constraints and increased stability of the protein in the subdenaturing region giving rise to gated entry of the photolyzed CO from the solvent. The rate for geminate rebinding does not show any decrease in the rate versus GdnHCl concentration plots. The values for the activation enthalpy for the CO dissociation reaction and the entropy loss relative to the native-state entropy, both measured as a function of GdnHCl concentration, indicate that the protein is indeed stabilized under subdenaturing conditions. Analyses of thermal unfolding transitions of myoglobin in the presence of different concentrations of GdnHCl indicate that the stability of this protein extracted from the linear free energy model is approximately 3-4 kcal mol(-1) less than the true stability. The results indicate the appropriateness of the denaturant binding model for the analysis of GdnHCl-induced unfolding data, and provide a value of 7.9 kcal mol(-1) as the true stability of the protein.

Guanidine-induced unfolding of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus

The unfolding induced by guanidine hydrochloride of the small protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus has been investigated by means of circular dichroism and fluorescence measurements. At neutral pH and room temperature the midpoint of the transition occurred at 4 M guanidine hydrochloride. Thermodynamic information was obtained by means of both the linear extrapolation model and the denaturant binding model, in the assumption of a two-state N⇔D transition. A comparison with thermodynamic data determined from the thermal unfolding of Sso7d indicated that the denaturant binding model has to be preferred. Finally, it is shown that Sso7d is the most stable against both temperature and guanidine hydrochloride among a set of globular proteins possessing a very similar 3D structure.

Guanidine-induced unfolding of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus

The unfolding induced by guanidine hydrochloride of the small protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus has been investigated by means of circular dichroism and fluorescence measurements. At neutral pH and room temperature the midpoint of the transition occurred at 4 M guanidine hydrochloride. Thermodynamic information was obtained by means of both the linear extrapolation model and the denaturant binding model, in the assumption of a two-state N⇔D transition. A comparison with thermodynamic data determined from the thermal unfolding of Sso7d indicated that the denaturant binding model has to be preferred. Finally, it is shown that Sso7d is the most stable against both temperature and guanidine hydrochloride among a set of globular proteins possessing a very similar 3D structure.

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.

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

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

A thermodynamic analysis of a family of small globular proteins: SH3 domains

The stability and folding thermodynamics of two SH3-domains, belonging to Fyn and Abl proteins, have been studied by scanning calorimetry and urea-induced unfolding. They undergo an essentially two-state unfolding with parameters similar to those of the previously studied α-spectrin SH3 domain. The correlations between the thermodynamic parameters (heat capacity increment, ΔC p,U , the proportionality factor, m , and the Gibbs energy, ΔG w 298) of unfolding and some integral structural parameters, such as polar and non-polar areas exposed upon domain denaturation, have been analyzed. The experimental data on ΔC p,U and the m- factor of the linear extrapolation model (LEM) obey the simple empirical correlations deduced elsewhere. The Gibbs energies calculated from the DSC data were compared with those found by fitting urea-unfolding curves to the LEM and the denaturant-binding model (DBM). The ΔG w 298 values found with DBM correlate better with the DSC data, while those obtained with LEM are systematically smaller. The systematic difference between the parameters calculated with LEM and DBM are explained by an inherent imperfection of the LEM.

New evidence for the denaturant binding model.

Denaturation with guanidine hydrochloride (GdnHCl) or urea is one of the primary ways of measuring the conformational stability of proteins and comparing the stability of mutant proteins. Despite the widespread use of these two denaturants to provide quantitative data for the free energies of unfolding, the mode of action of these agents is not well understood. We are not even certain whether the action of these agents on proteins is direct and can be regarded as ligand binding, or indirect and involves a change in the properties of solvent (water) in the presence of GdnHCl and urea. In this paper, an extensive kinetic study of the inhibition of ribonuclease A and papain by urea has been performed. The results suggest that the effect of urea on activities of these enzymes can be well described by the denaturant binding model. The binding constants of urea determined by the present method are nearly identical to that determined from a variety of different studies on model compounds and proteins.

Cold denaturation of ubiquitin

Temperature induced unfolding of bovine ubiquitin in solutions with different concentrations of guanidinium hydrochloride (GdmCl) has been measured using differential scanning calorimetry. It has been shown that at high concentrations of GdmCl the ubiquitin molecule can undergo both heat and cold induced denaturation. Analysis of the enthalpy of unfolding of ubiquitin in the presence of GdmCl shows a good agreement with the thermodynamic denaturant binding model. The unfolding Gibbs energy is found to change linearly with guanidine concentration up to zero denaturant concentration.

The Interaction of Micelles with Added Species and Its Similarity to the Denaturant Binding Model of Proteins

The effect of additives on micellar aggregation is treated using the same framework developed for macromolecular binding. This procedure, hinging on the pseudo-phase transition model, allows us to evaluate the binding constant of any added substance to a micellar aggregate as well as the number of particles bound per amphiphilic unit. The analysis of published literature data confirms that the model provides a general description of micellar aggregation in the presence of added ingredients, regardless of their ionic or non-ionic nature. Original data from our laboratory are well described by the suggested macromolecular binding approach. The dependence of experimental trends on micellization modifiers is also briefly discussed.

A Lysozyme Folding Intermediate Revealed by Solution X-ray Scattering

Equilibrium unfolding of hen egg lysozyme as a function of urea concentration at pH 2.9 has been studied by solution X-ray scattering. Differences in the unfolding transition are observed as monitored by the radius of gyration R g, and by far and near UV CD (circular dichroism) at 222 nm and 298 nm, respectively. This suggests the existence of a third unfolding species, in addition to the native and the unfolded states. A singular value decomposition (SVD) analysis was made of the scattering curves at different urea concentrations. This analysis shows clear evidence of a third basis component in the X-ray scattering curves, thus supporting the results of the R gand CD measurements. The denaturant binding model was employed to estimate the thermodynamic parameters of denaturation for the intermediate and unfolded states. Use of these parameters to refine the SVD analysis allows us to reconstruct a scattering profile for the pure intermediate state. Simplified partially folded models, based on the crystal structure of hen lysozyme, support a working model for the intermediate, whose structure may be correlated with that of the kinetic intermediate found in the refolding pathway studied by Dobson and coworkers.

Denaturation of horseradish peroxidase with urea and guanidine hydrochloride

Favourable effects of urea and guanidine hydrochloride (Gdn HCI) on solubilization of the polar, non-polar and peptide groups of horseradish peroxidase (HRP), an example of a globular protein, provide the driving force for unfolding of HRP, in a reversible two-state process. The intrinsic or conformational stability of HRP at various pH values and temperatures has been estimated by the linear extrapolation method (LEM), a denaturant binding model (DBM) and Tanford's model. There is good agreement between these methods. Tanford's model shows that urea interacts with non-polar groups to a greater extent than Gdn HCl does, whereas Gdn HCI interacts more effectively with the peptide groups of HRP.

Protein stability parameters measured by hydrogen exchange.

The hydrogen exchange (HX) rates of the slowest peptide group NH hydrogens in oxidized cytochrome c (equine) are controlled by the transient global unfolding equilibrium. These rates can be measured by one-dimensional nuclear magnetic resonance and used to determine the thermodynamic parameters of global unfolding at mild solution conditions well below the melting transition. The free energy for global unfolding measured by hydrogen exchange can differ from values found by standard denaturation methods, most notably due to the slow cis-trans isomerization of the prolyl peptide bond. This difference can be quantitatively calculated from basic principles. Even with these corrections, HX experiments at low denaturant concentration measure a free energy of protein stability that rises above the usual linear extrapolation from denaturation data, as predicted by the denaturant binding model of Tanford.

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.