Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.     

Thermodynamics, kinetics, and salt dependence of folding of YopM, a large leucine-rich repeat protein

Small globular proteins have many contacts between residues that are distant in primary sequence. These contacts create a complex network between sequence-distant segments of secondary structure, which may be expected to promote the cooperative folding of globular proteins. Although repeat proteins, which are composed of tandem modular units, lack sequence-distant contacts, several of considerable length have been shown to undergo cooperative two-state folding. To explore the limits of cooperativity in repeat proteins, we have studied the unfolding of YopM, a leucine-rich repeat (LRR) protein of over 400 residues. Despite its large size and modular architecture (15 repeats), YopM equilibrium unfolding is highly cooperative, and shows a very strong dependence on the concentration of urea. In contrast, kinetic studies of YopM folding indicate a mechanism that includes one or more transient intermediates. The urea dependence of the folding and unfolding rates suggests a relatively small transition state ensemble. As with the urea dependence, we have found an extreme dependence of the free energy of unfolding on the concentration of salt. This salt dependence likely results from general screening of a large number of unfavorable columbic interactions in the folded state, rather than from specific cation binding.     

Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased beta-sheet and reduced alpha-helix content relative to the native state. Urea-induced unfolding at 25 degrees C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15 degrees to 60 degrees C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25 degrees C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.     

Four-state equilibrium unfolding of an scFv antibody fragment

The conformational stability of a single-chain Fv antibody fragment against a hepatitis B surface antigen (anti-HBsAg scFv) has been studied by urea and temperature denaturation followed by fluorescence and circular dichroism. At neutral pH and low protein concentration, it is a well-folded monomer, and its urea and thermal denaturations are reversible. The noncoincidence of the fluorescence and circular dichroism transitions indicates the accumulation in the urea denaturation of an intermediate (I(1)) not previously described in scFv molecules. In addition, at higher urea concentrations, a red-shift in the fluorescence emission maximum reveals an additional intermediate (I(2)), already reported in the denaturation of other scFvs. The urea equilibrium unfolding of the anti-HBsAg scFv is thus four-state. A similar four-state behavior is observed in the thermal unfolding although the intermediates involved are not identical to those found in the urea denaturation. Global analysis of the thermal unfolding data suggests that the first intermediate displays substantial secondary structure and some well-defined tertiary interactions while the second one lacks well-defined tertiary interactions but is compact and unfolds at higher temperature in a noncooperative fashion. Global analysis of the urea unfolding data (together with the modeled structure of the scFv) provides insights into the conformation of the chemical denaturation intermediates and allows calculation of the N-I(1), I(1)-I(2), and I(2)-D free energy differences. Interestingly, although the N-D free energy difference is very large, the N-I(1) one, representing the "relevant" conformational stability of the scFv, is small.     

Calorimetric study of the thermal denaturation of beta-lactoglobulin in the presence of urea and phosphate ions]

The denaturation of beta-lactoglobulin in solution with different content of urea and phosphates has been studied calorimetrically. It has been shown that the increase of phosphate ion concentration in solution leads to an increase of beta-lactoglobulin stability, while increase of urea concentration leads to an opposite effect. The variation of these components in solution practically does not influence the value of the heat capacity increment of beta-lactoglobulin in the considered temperature region. Accordingly the denaturation enthalpy is a linear function of temperature whose slope does not differ for solution with urea concentration less than 4.4 M. However, the absolute value of denaturation enthalpy in these solutions at corresponding temperatures differs significantly due to the heat effect of additional urea solvation during transition to the denatured state. The latter leads to a decrease of the overall denaturation enthalpy and, as a result, a shift of the enthalpy plot to higher temperatures providing conditions for studying the thermodynamic and structural characteristics of the molecule in the cold denatured-state.     

Cooperative equilibrium transitions coupled with a slow annealing step explain the sharpness and hysteresis of collagen folding.

Heat and guanidinium-induced denaturation curves of collagen III and its fragments were fitted by theoretical models to explain the extreme sharpness and the hysteresis between unfolding and refolding. It was shown that a recently proposed kinetic model for collagen denaturation does not account for the observed steepness, with physically reasonable values of activation energy and frequency factors in the Arrhenius equation. The extreme slope, which amounts to 0.38 per centigrade for collagen III at the midpoint of its transition, can only be explained by a highly cooperative equilibrium model. The refolding curve is shifted to lower temperatures by 6 degrees C for collagen III and reversible unfolding matching the initial profile of the native protein is observed only after long-time annealing. A simple formalism is proposed by which experimental denaturation and refolding curves are quantitatively described. The transition proceeds via many cooperative steps with slightly different equilibrium constants for unfolding and refolding. Hysteresis and annealing are caused by very slow steps, which are probably connected with a rearrangement of misfolded regions. These slow steps disappear with decreasing size of collagen fragments and hysteresis is not found for collagen model peptides.     

Protein stability: functional dependence of denaturational Gibbs energy on urea concentration

Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data.     

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.     

Unfolding of the trp repressor from Escherichia coli monitored by fluorescence, circular dichroism and nuclear magnetic resonance

The denaturation of the trp repressor from Escherichia coli has been studied by fluorescence, circular dichroism and proton magnetic resonance spectroscopy. The dependences of the fluorescence emission of the two tryptophan residues on the concentration of urea are not identical. The dependence of the quenching of tryptophan fluorescence by iodide as a function of urea concentration also rules out a two-state transition. The circular dichroism at 222 nm decreases in two phases as urea is added. Normalised curves for different residues observed by 1H NMR also do not coincide, and require the presence of at least one stable intermediate. Analysis of the dependence of the denaturation curves on the concentration of protein indicate that the first transition is a partial unfolding of the dimeric repressor, resulting in a loss of about 25% of the helical content. The second transition is the dissociation and unfolding of the partially unfolded dimer. At high concentrations of protein (500 microM) about 73% of the repressor exists as the intermediate in 4 M urea. The apparent dissociation constant is about 10(-4) M; the subunits are probably strongly stabilised by the subunit interaction. The native repressor is stable up to at least 70 degrees C, whereas the intermediate formed at 4 M urea can be denatured reversibly by heating (melting temperature approximately 60 degrees C, delta H approximately 230 kJ/mol).     

Study of the structural properties of recombinant gamma-interferon by circular dichroism and differential scanning microcalorimetry]

Recombinant human gamma-interferon is dimeric in solution at pH 7-4 as revealed by analytical gel-filtration. It was shown by circular dichroism that decreasing pH to 5.0 does not affect the secondary and tertiary structures of gamma-interferon macromolecule. It was established that heat denaturation process of gamma-interferon obeys the two-state transition model and can be described as the first-order reversible reaction. Temperature dependence of the denaturation-renaturation rate constants was shown to be consistent with the Arrhenius law. The equilibrium value of the denaturation temperature was found. Effective enthalpy of denaturation was determined both by thermodynamic and kinetic approaches. The data obtained showed that in the pH range 7-4 the dimeric IFN-gamma structure may be considered as a single cooperative thermodynamic domain. Thus, it may be concluded that gamma-interferon dimerization is necessary for the existence of the corresponding tertiary structure of the macromolecule.     

Differential scanning calorimetry of the thermal denaturation of lactate dehydrogenase

1. Differential scanning calorimetry has been used to study the thermal denaturation of lactate dehydrogenase. At pH 7.0 in 0.1 M potassium phosphate buffer, only one transition was observed. Both the enthalpy of denaturation and the melting temperature are linear function of heating rate. The enthalpy is 430 kcal/mol and the melting temperature 61 degrees C at 0 degrees C/min heating rate. The ratio of the calorimetric heat to the effective enthalpy indicated that the denaturation is highly cooperative. Subunit association does not appear to significantly contribute to the enthalpy of denaturation. 2. Both cofactor and sucrose addition stabilized the protein against thermal denaturation. Pyruvate addition produced no changes. Only a small time-dependent destabilization was observed at low concentrations of urea. Large effects were observed in concentrated NaCl solutions and with sulfhydryl-modified lactate dehydrogenase.     

The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase.

In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.     

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.     

Decrease in stability of human albumin with increase in protein concentration

The stability (reflected in denaturation temperature, Td) of defatted human albumin monomer, monitored by differential scanning calorimetry, decreases with increasing protein concentration. This is shown to be compatible with a simple model in which reversible polymerization of denatured monomer promotes unfolding. This model also predicts an increase in transition cooperativity with decreasing protein concentration whereas experimentally cooperativity decreases because the rate of thermally induced polymerization of unfolded monomer is slow relative to the scan rate of the calorimeter. The denaturation of undefatted human albumin monomer, subsaturated with high affinity endogenous long-chain fatty acid (LCFA), was previously observed by differential scanning calorimetry to be a biphasic process. Td for the first endotherm, associated with the denaturation of LCFA-poor species, decreases with increasing protein concentration similar to that for defatted monomer whereas Td for the second endotherm, associated with denaturation of LCFA-rich species, is independent of concentration. The magnitude of the concentration dependence of Td relates directly to the extent of polymerization of denatured monomer, which decreases with increasing level of bound ligand. The bimodal thermogram observed for undefatted monomer persists upon simultaneous extrapolation of Td values to low concentration and low scan rate thereby demonstrating that this biphasic denaturation arising from ligand redistribution during denaturation is a true thermodynamic phenomenon and not an artifact of specific experimental conditions or the method used to induce denaturation.     

Solvent denaturation and stabilization 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 effects of solvents on protein stability. Folding is assumed to be driven by solvophobic interactions and opposed by conformational entropy. The solvent dependence of the solvophobic interactions is taken from transfer experiments of Nozaki and Tanford on amino acids into aqueous solutions of urea or guanidine hydrochloride (GuHCl). On the basis of the assumption of two pathways involving collapse and formation of a core, the theory predicts that increasing denaturant should lead to a two-state denaturation transition (i.e., there is a stable state along each path separated by a free energy barrier). The denaturation midpoint is predicted to occur at higher concentrations of urea than of GuHCl. At neutral pH, the radius of the solvent-denatured state should be much smaller than for a random-flight chain and increase with either denaturant concentration or number of polar residues in the chain. A question of interest is whether free energies of folding should depend linearly on denaturant, as is often assumed. The free energy is predicted to be linear for urea but to have some small curvature for GuHCl. Predicted slopes and exposed areas of the unfolded states are found to be in generally good agreement with experiments. We also discuss stabilizing solvents and compare thermal with solvent denaturation.     

Study of the physico-chemical properties of troponins I and T from the heart and skeletal muscles using protein fluorescence and calorimetry methods

Physico-chemical properties of troponin I and troponin T subunits from cardiac and skeletal muscles were studied, using intrinsic protein fluorescence and differential scanning microcalorimetry. The effects of temperature, pH, urea and ionic strength were analyzed. Similar skeletal and cardiac components were shown to possess similar properties. Alkali produced structural changes in both troponins I which seems to be initiated by deprotonation of histidyl side chains within the pH range of 6.5-9.0. An increase of pH from 9 to 12 results in alkaline denaturation transitions in both troponin I subunits, which might be due to deprotonation of tyrosyl side chains. A decrease of pH from 6 to 4 causes aggregation of both troponin T subunits. Cardiac troponin T is more stable to alkali and urea denaturation than the skeletal one. Heating up to 100 degrees C does not cause any cooperative denaturation transitions in troponins I and troponins T. These results suggest that cardiac and skeletal troponins I and troponins T possess a rather open, not highly ordered structure in solution.     

The contribution of electrostatic factors to the stabilization of the conformation of cytochrome c. Studies on the maleylated protein.

All the lysines of horse heart cytochrome c were maleylated yielding a low spin product. At room temperature and low salt concentration, this product lacked the 695 nm absorption band and showed tryptophan fluorescence and circular dichroic spectra typical of denatured cytochrome c. The 695 nm band and the native tryptophan fluorescence and circular dichroic spectra were restored by addition of salts, their effectiveness being dependent on the charge of the cation. On low salt concentration, the 695 nm band was also restored by lowering the temperature. Studies of the temperature dependence of the 695 nm band indicate that the thermal denaturation of maleylated cytochrome c occurs at temperatures 60-70 degrees C lower than in the native protein. This implies a destabilization of the native conformation by 5.6 kcal/mol; a similar value is evidenced by comparative urea denaturation studies on the native and modified proteins. The results confirm the assumption that the native conformation of cytochrome c is mostly determined by interactions involving internal residues.     

Kinetic inactivation study of mushroom tyrosinase: intermediate detection by denaturants

The unfolding and inhibition study of mushroom tyrosinase have been studied in the presence of different denaturants such as sodium dodecyl sulfate (SDS), guanidine hydrochloride (GdnHCl), and urea. The kinetic two-phase rate constants were commonly measured from semilogarithmic plots of the activity versus time, which resolved into two straight lines, indicating that the inactivation process consisted of fast and slow phases as a first-order reaction. This result also implied that transient partially folded intermediate existed during tyrosinase unfolding pathway. Mushroom tyrosinase had different behaviors to denaturants in regard with: noncooperative binding manner by SDS while cooperative interactions by GdnHCl and urea; in equilibrium state, SDS-micelle never completely inactivated enzyme activity while GdnHCl has single step denaturation and urea induced a typical transition-like process. Various kinetic parameters for each denaturant were calculated and the possible unfolding pathway scheme was discussed.     

Preferential interactions of urea with lysozyme and their linkage to protein denaturation

The interactions involved in the denaturation of lysozyme in the presence of urea were examined by thermal transition studies and measurements of preferential interactions of urea with the protein at pH 7.0, where it remains native up to 9.3 M urea, and at pH 2.0, where it undergoes a transition between 2.5 and 5.0 M urea. The destabilization of lysozyme by urea was found to follow the linear dependence on urea molar concentration, M(u), DeltaG(u)(o)=DeltaG(w)(o)-2.1 M(u), over the combined data, where DeltaG(u)(o) and DeltaG(w)(o) are the standard free energy changes of the N right harpoon over left harpoon D reaction in urea and water, respectively. Combination with the measured preferential binding gave the result that the increment of preferential binding, deltaGamma(23)=Gamma(23)(D)-Gamma(23)(N), is also linear in M(u). A temperature dependence study of preferential interactions permitted the evaluation of the transfer enthalpy, DeltaHmacr;(2,tr)(o), and entropy, DeltaSmacr;(2,tr)(o) of lysozyme from water into urea in both the native and denatured states. These values were found to be consistent with the enthalpy and entropy of formation of inter urea hydrogen bonds (Schellman, 1955; Kauzmann, 1959), with estimated values of DeltaHmacr;(2,tr)(o)=ca. -2.5 kcal mol(-1) and DeltaSmacr;(2,tr)(o)=ca. -7.0 e.u. per site. Analysis of the results led to the conclusion that the stabilization of the denatured form was predominantly by preferential binding to newly exposed peptide groups. Combination with the knowledge that stabilizing osmolytes act by preferential exclusion from peptide groups (Liu and Bolen, 1995) has led to the general conclusion that both the stabilization and destabilization of proteins by co-solvents are controlled predominantly by preferential interactions with peptide groups newly exposed on denaturation.     

Unfolding of the loggerhead sea turtle (Caretta caretta) myoglobin: A (1)H-NMR and electronic absorbance study

The effect of urea concentration on the backbone solution structure of the cyanide derivative of ferric Caretta caretta myoglobin (at pH 5.4) is reported. By addition of urea, sequential and long-range nuclear Overhauser effects (NOEs) are gradually lost. By using the residual NOE constraints to build the molecular model, a picture of the unfolding pathway was obtained. When the urea concentration is raised to 2.2 M, helices A and B appear largely disordered; helices C, D, and F loose structural constraints at 3.0 M urea. At urea concentration >6 M, the protein appears to be fully unfolded, including the GH hairpin and helix E stabilizing the prosthetic group. Reversible and cooperative denaturation isotherms obtained by following NOE peaks are considerably different from those obtained by monitoring electronic absorption changes. The reversible and cooperative urea-dependent folding-unfolding process of C. caretta myoglobin follows the minimum three-state mechanism N long left and right arrow X long left and right arrow D, where X represents a disordered globin structure (occurring at approximately 4 M urea) that still binds the heme.