Heat capacity and thermodynamic characteristics of denaturation and glass transition of hydrated and anhydrous proteins

Calorimetric measurements of absolute heat capacity have been performed for hydrated (11)S-globulin (0 < C(H(2)O) < 25%) and for lysozyme in a concentrated solution, both in the native and denatured states. The denaturation process is observed in hydrated and completely anhydrous proteins; it is accompanied by the appearance of heat capacity increment (Delta(N)(D)C(p)), as is the case for protein solutions. It has been shown that, depending on the temperature and water content, the hydrated denatured proteins can be in a highly elastic or glassy states. Glass transition is also observed in hydrated native proteins. It is found that the denaturation increment Delta(N)(D)C(p) in native protein, like the increment DeltaC(p) in denatured protein in glass transition at low water contents, is due to additional degrees of freedom of thermal motion in the protein globule. In contrast to the conventional notion, comparison of absolute C(p) values for hydrated denatured proteins with the C(p) values for denatured proteins in solution has indicated a dominant contribution of the globule thermal motion to the denaturation increment of protein heat capacity in solutions. The concentration dependence of denaturing heat absorption (temperature at its maximum, T(D), and thermal effect, DeltaQ(D)) and that of glass transition temperature, T(g), for (11)S-globulin have been studied in a wide range of water contents. General polymeric and specific protein features of these dependencies are discussed.     

Theory of cooperative transitions in protein molecules. I. Why denaturation of globular protein is a first-order phase transition

A theory of equilibrium denaturation of proteins is suggested. According to this theory, a cornerstone of protein denaturation is disruption of tight packing of side chains in protein core. Investigation of this disruption is the object of this paper. It is shown that this disruption is an "all-or-none" transition (independent of how compact is the denatured state of a protein and independent of the protein-solvent interactions) because expansion of a globule must exceed some threshold to release rotational isomerization of side chains. Smaller expansion cannot produce entropy compensation of nonbonded energy loss; this is the origin of a free-energy barrier (transition state) between the native and denatured states. The density of the transition state is so high that the solvent cannot penetrate into protein in this state. The results obtained in this paper make it possible to present in the following paper a general phase diagram of protein molecule in solution.     

Studies on protein folding, unfolding and fluctuations by computer simulation. I. The effect of specific amino acid sequence represented by specific inter-unit interactions.

A lattice model of proteins is introduced. "A protein molecule" is a chain of nown-intersecting units of a given length on the two-dimensional square lattice. The copolymeric character of protein molecules is incorporated into the model in the form of specificities of inter-unit interactions. This model proved most effective for studying the statistical mechanical characteristics of protein folding, unfolding and fluctuations. The specificities of inter-unit interactions are shown to be the primary factors responsible for the all-or-none type transition from native to denatured states of globular proteins. The model has been studied by the Monte Carlo method of Metropolis et al., which is now shown applied to approximately simulating a kinetic process. In the strong limit of the specificity of the inter-unit interaction the native conformation was reached in this method by starting from an extended conformation. The possible generalization and application of this method for finding the native conformation of proteins form their amino sequence are discussed.     

Cation-induced toroidal condensation of DNA studies with Co3+(NH3)6

The unfolding and refolding of Staphylococcus aureus penicillinase have been followed by urea-gradient electrophoresis. Unfolding of the native state proceeds by an all-or-none transition to fully unfolded protein, with no detectable accumulation of partially unfolded states. In contrast, refolding is complex and proceeds by very rapid, reversible formation of a partially folded state, H, which had been detected and characterized previously, as it is the most stable conformation at intermediate denaturant concentrations. At very low urea concentrations, a more compact conformational state was observed as a transient intermediate in refolding. There was little kinetic heterogeneity of the unfolded protein, as is normally observed with proteins containing proline residues.     

Domains in folding of model proteins.

By means of Monte Carlo simulation, we investigated the equilibrium between folded and unfolded states of lattice model proteins. The amino acid sequences were designed to have pronounced energy minimum target conformations of different length and shape. For short fully compact (36-mer) proteins, the all-or-none transition from the unfolded state to the native state was observed. This was not always the case for longer proteins. Among 12 designed sequences with the native structure of a fully compact 48-mer, a simple all-or-none transition was observed in only three cases. For the other nine sequences, three states of behavior-the native, denatured, and intermediate states-were found. The contiguous part of the native structure (domain) was conserved in the intermediate state, whereas the remaining part was completely unfolded and structureless. These parts melted separately from each other.     

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.     

Possibility for discriminating between two representative non two-state thermal unfolding models of proteins by DSC

Possible differences between two representative non two-state thermal unfolding mechanisms of protein are discussed concerning differential scanning calorimetry. Numerical simulations showed that, by DSC measurement, it is hard to discriminate between the independent model, which assumes independent unfolding domains in a protein, and the sequential model, which assumes intermediate(s) between native and denatured states, especially when values of molecular weight, denaturation enthalpy, and difference in denaturation temperature of each denaturation process are large. DSC curve analysis of Aspergillus niger glucoamylase based on these two models gave essentially the same thermodynamic parameters.     

Denatured increments of heat capacity in globular proteins and its connection with vitrification processes

Absolute values of heat capacity for some hydrated globular proteins have been studied by differential scanning calorimetry (DSC) method. It has been found that for the proteins with completely bound water, like in the case of protein solutions, the value of heat capacity of denatured proteins is higher than that prior to denaturation. Depending on temperature and humidity the denatured proteins can be either in high elastic or glass state. Specific heat capacities for these two states have the same values for all proteins and depend only on temperature with a characteristic increment of 0.55 J/g.K. at glass transition. The glass transitions were observed not only in denatured but also in native proteins. As it follows from our results, the main contribution to the heat capacity increment at denaturation is connected with the thermal motion in the protein globule which is in contrast with the commonly accepted ideas.     

Effects of sulphate and urea on the stability and reversible unfolding of beta-lactamase from Staphylococcus aureus. Implications for the folding pathway of beta-lactamase

The reversible denaturation by urea of beta-lactamase from Staphylococcus aureus was followed in the presence and absence of ammonium sulphate by circular dichroism studies, difference absorption spectroscopy and measurement of enzyme activity. The multiple unfolding and refolding transitions demonstrate the existence of a thermodynamically stable state of intermediate conformation in equilibrium with the native (N) and fully unfolded (U) states. Its physical properties show that it is identical to the state H found on denaturation by guanidinium chloride. State H is 10.1 (+/-1.5) kJ mol-1 less stable than the native state and 10.1 (+/-1.6) kJ mol-1 more stable than the unfolded state. Ammonium sulphate shifts both the N in equilibrium H and H in equilibrium U transitions to concentrations of urea higher by 5.3 M per mole of sulphate. It has markedly different effects on the thermodynamic stabilities of states N and H, making delta G'N-H, O and delta G'H-U, O more negative by 41 kJ mol and 20 kJ mole, respectively, per mole of ammonium sulphate. The change in equilibrium constant for the N-H transition is reflected almost exclusively in a dramatic change of the unfolding rate constant, which is decreased by a factor of 10(11) on addition of 1.4 M-sulphate. The presence of the substrate benzyl penicillin has little effect on the equilibria or kinetics of the N-H transition. The results are discussed in terms of the nature of the N-H transition and of the ordering of intermediate states on the folding pathway.     

The stability and folding process of amyloidogenic mutant human lysozymes.

Amyloid deposits are frequently formed by mutant proteins that have a lower stability than the wild-type proteins. Some reports, however, have shown that mutant-induced thermodynamic destabilization is not always a general mechanism of amyloid formation. To obtain a better understanding of the mechanism of amyloid fibril formation, we show in this study that equilibrium and kinetic refolding-unfolding reaction experiments with two amyloidogenic mutant human lysozymes (I56T and D67H) yield folding pathways that can be drawn as Gibbs energy diagrams. The equilibrium stabilities between the native and denatured states of both mutant proteins were decreased, but the degrees of instability were different. The Gibbs energy diagrams of the folding process reveal that the Gibbs energy change between the native and folding intermediate states was similar for both proteins, and also that the activation Gibbs energy change from the native state to the transition state decreased. Our results confirm that the tendency to favor the intermediate of denaturation facilitates amyloid formation by the mutant human lysozymes more than equilibrium destabilization between the native and completely denatured states does.     

Beyond Lumry-Eyring: an unexpected pattern of operational reversibility/irreversibility in protein denaturation

We have found that, contrary to naïve intuition, the degree of operational reversibility in the thermal denaturation of lipase from Thermomyces lanuginosa (an important industrial enzyme) in urea solutions is maximum when the protein is heated several degrees above the end of the temperature‐induced denaturation transition. Upon cooling to room temperature, the protein seems to reach a state with enzymatic activity similar to that of the initial native state, but with higher denaturation temperature and radically different behavior in terms of susceptibility to irreversible denaturation. These results show that patterns of operational reversibility/irreversibility in protein denaturation may be more complex than the often‐taken‐for‐granted, two‐situation classification (reversible vs. irreversible). Furthermore, they are consistent with the possibility of existence of different native or native‐like states separated by high kinetic barriers under native conditions and they suggest experimental procedures to reach and study such “alternative” native states. Proteins 2008. © 2007 Wiley‐Liss, Inc.     

Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy

To elucidate the structure of denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra from 260 to 172 nm of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and the acid-, cold-, and heat-denatured states, using a synchrotron-radiation VUVCD spectrophotometer. The circular dichroism spectra of proteins fully unfolded by guanidine hydrochloride (GdnHCl) were also measured down to 197 nm for comparison. These denatured proteins exhibited characteristic VUVCD spectra that reflected a considerable amount of residual secondary structures. The contents of alpha-helices, beta-strands, turns, poly-L-proline type II (PPII), and unordered structures were estimated for each denatured state of the three proteins using the SELCON3 program with Protein Data Bank data and the VUVCD spectra of 31 reference proteins reported in our previous study. Based on these contents, the characteristics of the four types of denaturation were discussed for each protein. In all types of denaturation, a decrease in alpha-helices was accompanied by increases in beta-strands, PPII, and unordered structures. About 20% beta-strands were present even in the proteins fully unfolded by GdnHCl in which beta-sheets should be broken. From these results, we propose that denatured proteins constitute an ensemble of residual alpha-helices and beta-sheets, partly unfolded (or distorted) alpha-helices and beta-strands, PPII, and unordered structures.     

Mechanisms of temporary inhibition in Streptomyces subtilisin inhibitor induced by an amino acid substitution, tryptophan 86 replaced by histidine

Just one amino acid substitution (Trp86 replaced by His), which is more than 30 A away from the reactive site, changed the inhibitor, Streptomyces subtilisin inhibitor (SSI), into a temporary inhibitor without a change in the inhibition constant. When the inhibitor was in excess of subtilisin BPN', the wild-type SSI was stable under protease attack, while the mutant inhibitor was hydrolyzed to peptide fragments in an all-or-none manner. The mechanism of this temporary inhibition induced by the amino acid substitution was studied on the basis of structural, thermodynamic, and kinetic data obtained by a combined use of NMR, hydrogen-deuterium exchange, differential scanning calorimetry, and gel filtration HPLC. The mutation did not induce major structural changes, and in particular, the structure of the enzyme-binding region was virtually unaffected. The denaturation temperature of SSI, however, was decreased by 10 deg upon mutation, although it still remained a thermostable protein with a denaturation temperature of 73 degrees C. Furthermore, the activation enthalpy for denaturation was reduced dramatically, to half that of the wild type. When the mutated SSI is present in excess of the enzyme, the proteolysis followed first-order reaction kinetics with respect to the total concentration of the mutated SSI molecules present. From these combined results, we conclude that the proteolysis proceeds not through the native form of the inhibitor in the inhibitor-enzyme complex but through the denatured (unfolded) form of the inhibitor whose fraction is increased by the mutation. This conclusion states that the necessary condition for being a serine protease inhibitor lies not only in the design of the reactive site structure that is highly resistant to protease attack but also in the suppression of such structural fluctuation that brings about cooperative denaturation. In contrast, when the protease existed in excess of the mutated inhibitor, the proteolysis reaction was accelerated by more than 2 orders of magnitude. Furthermore, the reaction occurred even in the wild-type SSI at a comparable rate as in the mutated protein. This indicates that in the enzyme excess case another, more efficient digestion mechanism involving fluctuation within the native manifold of the inhibitor dominates.     

Comparative Study of Thermostability and Structure of Close Homologues - Barnase and Binase

Abstract

Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase in the pH region 2–6 have been determined. The barnase heat denaturation (pH 2.85.5) proceeds according to the “all-or-none” principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 °C at pH 5 to 7 °C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5–5.5, but as far as pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp94by the His 18 residue, absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moments distribution, have been found.     

A simple lattice model that exhibits a protein-like cooperative all-or-none folding transition

In a recent paper (D. Gront et al., Journal of Chemical Physics, Vol. 115, pp. 1569, 2001) we applied a simple combination of the Replica Exchange Monte Carlo and the Histogram methods in the computational studies of a simplified protein lattice model containing hydrophobic and polar units and sequence-dependent local stiffness. A well-defined, relatively complex Greek-key topology, ground (native) conformations was found; however, the cooperativity of the folding transition was very low. Here we describe a modified minimal model of the same Greek-key motif for which the folding transition is very cooperative and has all the features of the "all-or-none" transition typical of real globular proteins. It is demonstrated that the all-or-none transition arises from the interplay between local stiffness and properly defined tertiary interactions. The tertiary interactions are directional, mimicking the packing preferences seen in proteins. The model properties are compared with other minimal protein-like models, and we argue that the model presented here captures essential physics of protein folding (structurally well-defined protein-like native conformation and cooperative all-or-none folding transition).     

Nonideality and protein thermal denaturation

We studied the thermal denaturation of eglin c by using CD spectropolarimetry and differential scanning calorimetry (DSC). At low protein concentrations, denaturation is consistent with the classical two-state model. At concentrations greater than several hundred microM, however, the calorimetric enthalpy and the midpoint transition temperature increase with increasing protein concentration. These observations suggested the presence of intermediates and/or native state aggregation. However, the transitions are symmetric, suggesting that intermediates are absent, the DSC data do not fit models that include aggregation, and analytical ultracentrifugation (AUC) data show that native eglin c is monomeric. Instead, the AUC data show that eglin c solutions are nonideal. Analysis of the AUC data gives a second virial coefficient that is close to values calculated from theory and the DSC data are consistent with the behavior expected for nonideal solutions. We conclude that the concentration dependence is caused by differential nonideality of the native and denatured states. The nondeality arises from the high charge of the protein at acid pH and is exacerbated by low buffer concentrations. Our conclusion may explain differences between van't Hoff and calorimetric denaturation enthalpies observed for other proteins whose behavior is otherwise consistent with the classical two-state model.     

Stability of proteins in the presence of polyols estimated from their guanidinium chloride-induced transition curves at different pH values and 25 degrees C

We have recently concluded from the heat-induced denaturation studies that polyols do not affect deltaG(D) degrees (the Gibbs free energy change (deltaG(D)) at 25 degrees C) of ribonuclease-A and lysozyme at physiological pH and temperature, and their stabilizing effect increases with decrease in pH. Since the estimation of deltaG(D) degrees of proteins from heat-induced denaturation curves requires a large extrapolation, the reliability of this procedure for the estimation of deltaG(D) degrees is always questionable, and so are conclusions drawn from such studies. This led us to measure deltaG(D) degrees of ribonuclease-A and lysozyme using a more accurate method, i.e., from their isothermal (25 degrees C) guanidinium chloride (GdmCl)-induced denaturations. We show that our earlier conclusions drawn from heat-induced denaturation studies are correct. Since the extent of unfolding of heat- and GdmCl-induced denatured states of these proteins is not identical, the extent of stabilization of the proteins by polyols against heat and GdmCl denaturations may also differ. We report that in spite of the differences in the structural nature of the heat- and GdmCl-denatured states of each protein, the extent of stabilization by a polyol is same. We also report that the functional dependence of deltaG(D) of proteins in the presence of polyols on denaturant concentration is linear through the full denaturant concentration range. Furthermore, polyols do not affect the secondary and tertiary structures of the native and GdmCl-denatured states.     

Revealing the nature of the native state ensemble through cold denaturation

Recent advances in NMR methodology have enabled the structural analysis of proteins at temperatures far below the freezing point of water, thus opening a window to the cold denaturation process. Although the phenomenon of cold denaturation has been known since the mid-1970s, the freezing point of water has prevented detailed and structurally resolved studies without application of additional significant perturbations of the protein ensemble. As a result, the cold-denatured state and the process of cold denaturation have gone largely unstudied. Here, the structural and thermodynamic basis of cold denaturation is explored with emphasis placed on the insights that are uniquely ascertained from low-temperature studies. It is shown that the noncooperative cold-induced unfolding of protein results in the population of partially folded states that cannot be accessed by other techniques. The structurally resolved view of the cold denaturation process therefore can provide direct access to the cooperative substructures within the protein molecule and provide an unprecedented structurally resolved picture of the states that comprise the native state ensemble.