Thermodynamic stability of porcine beta-lactoglobulin. A structural relevance.

The proposed biological function of beta-lactoglobulins as transporting proteins assumes a binding ability for ligands and high stability under the acidic conditions of the stomach. This work shows that the conformational stability of nonruminant porcine beta-lactoglobulin (BLG) is not consistent with this hypothesis. Thermal denaturation of porcine BLG was studied by high-sensitivity differential scanning calorimetry within the pH range 2.0-10.0. Dependences of the denaturation temperature and enthalpy on pH were obtained, which reveal a substantial decrease in both parameters in acidic and basic media. The denaturation enthalpy follows a linear dependence on the denaturation temperature. The slope of this line is 9.4 +/- 0.6 kJ.mol-1. K-1,which is close to the denaturation heat capacity increment DeltadCp = 9.6 +/- 0.5 kJ.mol-1.K-1, determined directly from the thermograms. At pH 6.25 the denaturation temperatures of porcine and bovine BLG coincide, at 83.2 degrees C. At this pH the denaturation enthalpy of porcine BLG is 300 kJ.mol-1. The denaturation transition of porcine BLG was shown to be reversible at pH 3.0 and pH 9.0. The transition profile at both pH values follows the two-state model of denaturation. Based on the pH-dependence of the transition temperature and the linear temperature dependence of the transition enthalpy, the excess free energy of denaturation, DeltadGE, of porcine BLG was calculated as a function of pH and compared with that of bovine BLG derived from previously reported data. The pH-dependence of DeltadGE is analysed in terms of the contributions of side-chain H-bonds to the protein stability. Interactions stabilizing native folds of porcine and bovine BLG are discussed.     

Solvent denaturation of globular proteins: unfolding by the monoalkyl- and dialkyl-substituted formamides and ureas.

The effects of the monoalkyl and dialkyl-substituted formamide series of denaturants on the native conformation of sperm whale myoglobin, horse heart cytochrome c, and Glycera dibranciata (single chain) hemoglobin have been investigated by spectral measurements in the Soret region (409 and 422 nm) and optical rotation measurements (265nm). The effectiveness of these two classes of protein denaturants is similar to the other straight-chain compounds of the urea, amide, and alcohol classes, examined in previous investigations from our laboratory. Their denaturing effectiveness is found to increase with increasing chain length or hydrocarbon content of the substituent alkyl groups. Application of the Peller and Flory equation to the denaturation data of the formamides shows that both the polar and the nonpolar group contributions to the protein-denaturant interactions have to be taken into account in order to correctly predict the observed denaturation midpoints. Additivity of the hydrophobic, KH?, and the polar, Kp, group contributions to the binding constants, KB = nKH? + Kp, with n = 1 or 2 for the mono- of the di-alkyl substituted denaturants gave best account of the experimental data. The KH? values used were based on free energy transfer data of various alkyl groups or the Scheraga-Nemethy theory of hydrophobic bonding. The assumption of group contributions of the denaturant to KB were also applied to the denaturation data of the unsubstituted amides and some examples of the monoalkyl and symmetrically substituted dialkyl ureas, taken from the literature.     

pH dependence of listeriolysin O aggregation and pore-forming ability

Listeriolysin O (LLO) is the major factor implicated in the escape of Listeria monocytogenes from the phagolysosome. It is the only representative of cholesterol-dependent cytolysins that exhibits pH-dependent activity. Despite intense studies of LLO pH-dependence, this feature of the toxin still remains incompletely explained. Here we used fluorescence and CD spectroscopy to show that the structure of LLO is not detectably affected by pH at room temperature. We observed slightly altered haemolytic and permeabilizing activities at different pH values, which we relate to reduced binding of LLO to the lipid membranes. However, alkaline pH and elevated temperatures caused rapid denaturation of LLO. Aggregates of the toxin were able to bind Congo red and Thioflavin T dyes and were visible under transmission electron microscopy as large, amorphous, micrometer-sized assemblies. The aggregates had the biophysical properties of amyloid. Analytical ultracentrifugation indicated dimerization of the protein in acidic conditions, which protects the protein against premature denaturation in the phagolysosome, where toxin activity takes place. We therefore suggest that LLO spontaneously aggregates at the neutral pH found in the host cell cytosol and that this is a major mechanism of LLO inactivation.     

Protein interactions with urea and guanidinium chloride. A calorimetric study.

The interaction of urea and guanidinium chloride with proteins has been studied calorimetrically by titrating protein solutions with denaturants at various fixed temperatures, and by scanning them with temperature at various fixed concentrations of denaturants. It has been shown that the observed heat effects can be described in terms of a simple binding model with independent and similar binding sites. Using the calorimetric data, the number of apparent binding sites for urea and guanidinium chloride have been estimated for three proteins in their unfolded and native states (ribonuclease A, hen egg white lysozyme and cytochrome c). The intrinsic and total thermodynamic characteristics of their binding (the binding constant, the Gibbs energy, enthalpy, entropy and heat capacity effect of binding) have also been determined. It is found that the binding of urea and guanidinium chloride by protein is accompanied by a significant decrease of enthalpy and entropy. At all concentrations of denaturants the enthalpy term slightly dominates the entropy term in the Gibbs energy function. Correlation analysis of the number of binding sites and structural characteristics of these proteins suggests that the binding sites for urea and guanidinium chloride are likely to be formed by several hydrogen bonding groups. This type of binding of the denaturant molecules should lead to a significant restriction of conformational freedom within the polypeptide chain. This raises a doubt as to whether a polypeptide chain in concentrated solutions of denaturants can be considered as a standard of a random coil conformation.     

Fluorimetric characterization and influence of Cu2+ binding on the fluorescence of inorganic pyrophosphatase from baker's yeast.

The denaturation characteristics of inorganic pyrophosphatase from baker's yeast and the interaction with Cu2+ were investigated with fluorimetric methods. The position of the fluorescence emission spectrum with a maximum at 328 nm together with a quantum yield of 0.12 led to the conclusion that most of the tryptophan residues of the protein are buried in nonpolar inner regions of the molecule. The contribution of the tyrosine residues to the fluorescence of pyrophosphatase is only about 7%. Denaturation of the protein with denaturants or changes of the pH value cause a red shift of the fluorescence emission maximum. In the presence of Cu2+ ions a fluorescence quenching is observed. Thereby, a specific binding of one Cu2+ per subunit may be distinguished from further unspecific Cu2+ binding. The Cu2+ binding to the latter sites shows a time dependence according to a slow, reversible exposure of additional binding sites. This time dependent binding characteristics was also verified by following the free Cu2+ concentration with the fluorescent "metal indicator" epsilon-ADP.     

Free energy changes in denaturation of ribonuclease A by mixed denaturants. Effects of combinations of guanidine hydrochloride and one of the denaturants lithium bromide, lithium chloride, and sodium bromide

The denaturation of ribonuclease A by guanidine hydrochloride, lithium bromide, and lithium chloride and by mixed denaturants consisting of guanidine hydrochloride and one of the denaturants lithium chloride, lithium bromide, and sodium bromide was followed by difference spectral measurements at pH 4.8 and 25 degrees C. Both components of mixed denaturant systems enhance each other's effect in unfolding the protein. The effect of lithium bromide on the midpoint of guanidine hydrochloride denaturation transition is approximately the sum of the effects of the constituent ions. For all the mixed denaturants tested, the dependence of the free energy change on denaturation is linear. The conformational free energy associated with the guanidine hydrochloride denaturation transition in water is 7.5 +/- 0.1 kcal mol-1, and it is unchanged in the presence of low concentrations of lithium bromide, lithium chloride, and sodium bromide which by themselves are not concentrated enough to unfold the protein. The conformational free energy associated with the lithium bromide denaturation transition in water is 11.7 +/- 0.3 kcal mol-1, and it is not affected by the presence of low concentrations of guanidine hydrochloride which by themselves do not disrupt the structure of native ribonuclease A.     

Conformational plasticity of cryptolepain: accumulation of partially unfolded states in denaturants induced equilibrium unfolding

pH and chemical denaturant dependent conformational changes of a serine protease cryptolepain from Cryptolepis buchanani are presented in this paper. Activity measurements, near UV, far UV CD, fluorescence emission spectroscopy, and ANS binding studies have been carried out to understand the folding mechanism of the protein in the presence of denaturants. pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. The preliminary biophysical study on cryptolepain shows that major elements of secondary structure are beta-sheets. Under neutral conditions the enzyme was stable in urea while GuHCl-induced equilibrium unfolding was cooperative. Cryptolepain shows little ANS binding even under neutral conditions due to more hydrophobicity of beta-sheets. Multiple intermediates were populated during the pH-induced unfolding of cryptolepain. Temperature-induced denaturation of cryptolepain in the molten globule like state is non-cooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts, possibly domains, in the molecular structure of cryptolepain, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of A state (molten globule state) of cryptolepain is unique, as lower concentration of denaturant, not only induces structure but also facilitate transition from one molten globule like state (MG(1)) into another (MG(2)). The increase of pH drives the protein into alkaline denatured state characterized by the absence of any ANS binding. GuHCl- and urea-induced unfolding transition curves at pH 12.0 were non-coincidental indicating the presence of an intermediate in the unfolding pathway.     

Design and characteristics of a stable protein scaffold for specific binding based on variable lymphocyte receptor sequences

Variable lymphocyte receptors (VLRs) serve as antigen binding proteins in jawless vertebrates. Their relatively low molecular weight makes VLRs an interesting alternative to antibodies in biotechnological applications. A typical VLR comprises several unique motifs called leucine-rich repeats (LRRs). Using consensus approach we designed a novel VLR protein (called dVLR) containing six LRR repeats based on a sea lamprey receptor sequence. The designed protein was expressed in Escherichia coli in a soluble, native form and showed very favorable biophysical properties. Recombinant dVLR is monomeric in solution and preserves its secondary structure within the pH range 3.0 to 11.0 and tertiary structure between pH 4.0 and 10.0. It undergoes reversible thermal denaturation in a broad pH range (4.0 to 10.0). The maximal denaturation temperature of 73.9°C is observed at pH 6.0, 0.3M NaCl. Chemical denaturation of dVLR at pH 7.5 is a cooperative two-state process with a midpoint at 3.3M GdmCl and a very high free energy change of unfolding in the absence of denaturant equal to 14.1kcal/mol. The biophysical properties of dVLR make it highly suitable for biotechnological applications such as generation of specific ligand-binding molecules.     

Use of protein unfolding studies to determine the conformational and dimeric stabilities of HIV-1 and SIV proteases.

The free energies of dimer dissociation of the retroviral proteases (PRs) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were determined by measuring the effects of denaturants on the protein fluorescence upon the unfolding of the enzymes. HIV-1 PR was more stable to denaturation by chaotropes and extremes of pH and temperature than SIV PR, indicating that the former enzyme has greater conformational stability. The urea unfolding curves of both proteases were sigmoidal and single phase. The midpoints of the transition curves increased with increasing protein concentrations. These data were best described by and fitted to a two-state model in which folded dimers were in equilibrium with unfolded monomers. This denaturation model conforms to cases in which protein unfolding and dimer dissociation are concomitant processes in which folded monomers do not exist [Bowie, J. U., & Sauer, R. T. (1989) Biochemistry 28, 7140-7143]. Accordingly, the free energies of unfolding reflect the stabilities of the protease dimers, which for HIV-1 PR and SIV PR were, respectively, delta GuH2O = 14 +/- 1 kcal/mol (Ku = 39 pM) and 13 +/- 1 kcal/mol (Ku = 180 pM). The binding of a tight-binding, competitive inhibitor greatly stabilized HIV-1 PR toward urea-induced unfolding (delta GuH2O = 19.3 +/- 0.7 kcal/mol, Ku = 7.0 fM). There were also profound effects caused by adverse pH on the protein conformation for both HIV-1 PR and SIV PR, resulting in unfolding at pH values above and below the respective optimal ranges of 4.0-8.0 and 4.0-7.0     

Heat,Acid and Chemically Induced Unfolding Pathways,Conformational Stability and Structure-Function Relationship in Wheat α-Amylase

Wheat α-amylase, a multi-domain protein with immense industrial applications, belongs to α+β class of proteins with native molecular mass of 32 kDa. In the present study, the pathways leading to denaturation and the relevant unfolded states of this multi-domain, robust enzyme from wheat were discerned under the influence of temperature, pH and chemical denaturants. The structural and functional aspects along with thermodynamic parameters for α-amylase unfolding were probed and analyzed using fluorescence, circular dichroism and enzyme assay methods. The enzyme exhibited remarkable stability up to 70°C with tendency to aggregate at higher temperature. Acid induced unfolding was also incomplete with respect to the structural content of the enzyme. Strong ANS binding at pH 2.0 suggested the existence of a partially unfolded intermediate state. The enzyme was structurally and functionally stable in the pH range 4.0–9.0 with 88% recovery of hydrolytic activity. Careful examination of biophysical properties of intermediate states populated in urea and GdHCl induced denaturation suggests that α-amylase unfolding undergoes irreversible and non-coincidental cooperative transitions, as opposed to previous reports of two-state unfolding. Our investigation highlights several structural features of the enzyme in relation to its catalytic activity. Since, α-amylase has been comprehensively exploited for use in a range of starch-based industries, in addition to its physiological significance in plants and animals, knowledge regarding its stability and folding aspects will promote its biotechnological applications.     

Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: spectroscopic description of the native, intermediate, and unfolded states

Ervatamin A, a cysteine proteases from Ervatamia coronaria, has been used as model system to examine structure-function relationship by equilibrium unfolding methods. Ervatamin A belongs to alpha+beta class of proteins and exhibit stability towards temperature and chemical denaturants. Acid induced unfolding of ervatamin A was incomplete with respect to the structural content of the enzyme. Between pH 0.5 and 2.0, the enzyme is predominantly in beta-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (I(A) state). Surprisingly, high concentrations of GuHCl required to unfold this state and the transition mid points GuHCl induced unfolding curves are significantly higher. GuHCl induced unfolding of ervatamin A at pH 3.0 as well as at pH 4.0 is complex and cannot be satisfactorily fit to a two-state model for unfolding. Besides, a strong ANS binding to the protein is observed at low concentration of GuHCl, indicating the presence of intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8M) the enzyme retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to urea unfolding at pH 3.0 and below. Urea induced unfolding of ervatamin A at pH 3.0 is cooperative and the transitions curves obtained by different probes are and non-coincidental. Temperature denaturation of ervatamin A in I(A) state is non-cooperative, contrary to the cooperativity seen with native protein, suggesting the presence of two parts in the molecular structure of ervatamin A may be domains, with different stability that unfolds in steps. Careful inspection of biophysical properties of intermediate states populated in urea and GuHCl (I(UG) state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (I(A) state) identified at pH approximately 1.5 are different from those of the I(UG) state.     

Thermal stability of fatty acid-serum albumin complexes studied by differential scanning calorimetry.

Differential scanning calorimetry has been used to study the thermal stability of bovine serum albumin as affected by binding of fatty acids (lauric acid and stearic acid) and sodium dodecyl sulfate (SDS). All the ligands stabilized the protein molecules in a similar manner, but to different levels. A maximum increase in denaturation temperature of 30 degrees C was obtained with lauric acid. The thermograms indicate the presence of several ligand-albumin complexes having different heat stabilities. Variations in pH in 0.9% NaCl affected the heat stability of both ligand-poor and ligand-rich albumin, the former being more sensitive to variations in pH within the physiological range. Variations in NaCl concentration affected the thermal stabilities at neutral pH, expecially at low salt concentrations. While ligand-rich albumin was somewhat destabilized by increasing NaCl concentrations, ligand-poor albumin was strongly stabilized. The potential use of differential scanning calorimetry in ligand-albumin research is discussed.     

Theoretical studies of the electrostatic interactions in aspartic proteinases, intramolecular interactions in pepsin and penicillopepsin

A semi-empirical approach has been used to estimate the intramolecular electrostatic interactions in pepsin and penicillopepsin. The pH-dependence of the free energy electrostatic term was calculated, and the pH-dependence of the domain interactions has been estimated. As it was shown, the contribution of electrostatic interactions is rather small for the stabilization of the native structure. At the same time the electrostatic repulsion between domains increases with the increase of pH. The later can be the cause of the alkaline denaturation of pepsin and domain mobility.     

Thermodynamic investigations of proteins. II. Calorimetric study of lysozyme denaturation by guanidine hydrochloride.

The thermodynamic parameters of the denaturation of lysozyme are determined at various temperatures (25-60 degrees C) by isothermal calorimetric titrations with guanidine hydrochloride (GuHCl) and by scanning calorimetry in the presence of GuHCl. An approach for the determination of the enthalpy of preferential binding of GuHCl is proposed. It has been shown from GuHCl denaturation experiments that the net enthalpies of denaturation and the denaturational change in the heat capacity of protein can be obtained if preferential binding is taken into consideration. These results are nearly the same as in the case of thermal denaturation in the absence of denaturants. It is concluded that the states of both heat- and GuHCl-denatured lysozyme are thermodynamically indistinguishable.     

Structural characterization of protein-denaturant interactions: crystal structures of hen egg-white lysozyme in complex with DMSO and guanidinium chloride

A variety of physico-chemical methods employ chemical denaturants to unfold proteins, and study different biophysical processes involved therein. Chemical denaturants are believed to induce unfolding by stabilizing the unfolded state of proteins over the folded state, either macroscopically or through specific interactions. In order to characterize the nature of specific interactions between proteins and denaturants, we have solved crystal structures of hen egg-white lysozyme complexed with denaturants, and report here dimethyl sulfoxide and guanidinium chloride complexes. The dimethyl sulfoxide molecules and guanidinium ions were seen to bind the protein at specific sites and were involved in characteristic interactions. They share a major binding site between them, the C site in the sugar binding cleft of the enzyme. Although the overall conformations of the complexes were very similar to the native structure, spectacular conformational changes were seen to occur locally. Temperature factors were also seen to drop dramatically in the local regions close to the denaturant binding sites. An interesting observation of the present study was the generation of a sodium ion binding site in hen egg-white lysozyme in the presence of denaturants, which was hitherto unknown in any of the other lysozyme structures solved so far. Loss of some of the crucial side chain-main chain interactions may form the initial events in lysozyme unfolding.     

Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 1. CD and DSC studies

Cold denaturation and heat denaturation of the protein Streptomyces subtilisin inhibitor (SSI) were studied in the pH range 1.84-3.21 and in the temperature range -3-70 degrees C by circular dichroism and scanning microcalorimetry. The native structure of the protein was apparently most stabilized at about 20 degrees C and was denatured upon heating and cooling from this temperature. Each denaturation was reversible and cooperative, proceeding in two-state transitions, that is, from the native state to the cold-denatured state or from the native state to the heat-denatured state. The two denatured states, however, were not perfect random-coiled structures, and they differed from each other, indicating that there exist three states in this temperature range, i.e., cold denatured, native, and heat denatured. The difference between the cold and heat denaturations was indicated first by circular dichroism. The isodichroic point for the transition from the native state to the cold-denatured state was different from that from the native state to the heat-denatured state in the pH range between 3.21 and 2.45. Moreover, molar ellipticity for the cold-denatured state was different from that of the heat-denatured state, and the transition from the former to the latter was observed at pH values below 2. Values of van't Hoff enthalpies from the native state to the heat-denatured state at pH values between 3.21 and 2.45 were obtained by curve fitting of the CD data, and delta Cp = 1.82 (+/- 0.11) [kcal/(mol.K)] was obtained from the linear plot of the enthalpies against temperature. The parameters obtained from the heat denaturation studies gave curves for delta G zero which were not in agreement with the experimental data in the cold denaturation region when extrapolated to the low temperature. Moreover, the value of the apparent delta Cp for the cold denaturation in the pH range 3.03-2.45 was estimated to be different from that for the heat denaturation, indicating that the mechanism of the cold denaturation of SSI is different from a simple cold denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calorimetric study of the formation and melting of thermotropic gel in solutions of globular proteins

Scanning calorimetry has been used for studying lysozyme water solutions of different buffer molarity (mu = 0.5 divided by 1.0) and concentrations (c = 1.5 divided by 25%) at pH 2.0. It is shown that an additional high temperature maximum (HTM) can be observed on the heating curves for lysozyme solutions during irreversible denaturation. Calorimetric and rheological studies under identical heating conditions have shown that aggregation of protein during denaturation leads to the formation of the thermotropic gel. Further increase of temperature brings up the melting of this gel which results in the appearance of HTM on thermograms. Slow cooling of lysozyme gel melt leads to its reconstruction which results in the appearance of exothermic maximum on the corresponding thermograms.     

Ligand-induced biphasic protein denaturation

The results of a thermodynamic calculation of the excess heat capacity that is based on experimental observations and that incorporates the effects of ligand binding on the two-state, thermal denaturation of a protein are presented. For a protein with a single-binding site on the native species and at subsaturating concentrations of ligand, bimodal or unimodal thermograms were computed merely by assuming a larger or smaller ligand association constant, respectively. The calculated thermograms for this simplified case show the salient features of those observed by differential scanning calorimetry for defatted human albumin monomer in the absence and presence of three ligands for which the protein has higher, intermediate, and lower affinity (Shrake, A., and Ross, P. D. (1988) J. Biol. Chem. 263, 15392-15399). The computation demonstrates that biphasic unfolding can result from a significant increase in the free energy of denaturation (and the transition temperature) during the course of unfolding due to a substantial increase in free ligand concentration caused by the release of bound ligand by denaturing protein. Such ligand-induced biphasic denaturation does not relate to macromolecular substructure but derives from a perturbation, during unfolding, of the ligand binding equilibrium, which is coupled to the equilibrium between the folded and unfolded protein species. Thus, this bimodality is not limited to thermally induced unfolding but is operative independent of the means used to effect denaturation and therefore must be considered when studying any macromolecular folding/unfolding reaction in the presence of ligand.