Denaturation Behavior Research Articles

Kinetic and conformational effects of lysine substitutions for arginines 35 and 87 in the active site of staphylococcal nuclease

The high-resolution X-ray crystal structure of staphylococcal nuclease (SNase) suggests that the guanidinium groups of Arg 35 and Arg 87 participate as electrophilic catalysts in the attack of water on the substrate phosphodiester. Both arginine residues have been replaced with "conservative" lysine residues so that both the importance of these residues in catalysis and the effect of changes in electrostatic interactions on active site conformation can be assessed. The catalytic efficiencies of R35K and R87K are decreased by factors of 10(4) and 10(5) relative to wild-type SNase, with R87K showing a very significant reduction in its affinity for both DNA substrate and the competitive inhibitor thymidine 3',5'-bisphosphate (pdTp). The thermal denaturation behavior of both mutant enzymes differs from that of wild type both in the absence and in the presence of the active site ligands Ca2+ and pdTp. Both the 1H NMR chemical shifts and interresidue nuclear Overhauser effects (NOEs) of residues previously assigned to be in the hydrophobic core of SNase are altered in R35K and R87K. These observations, similar to those recently reported by our laboratories for substitutions for Glu 43 [Hibler, D. W., Stolowich, N. J., Reynolds, M. A., Gerlt, J. A., Wilde, J. A., & Bolton, P. H. (1987) Biochemistry 26, 6278; Wilde, J. A., Bolton, P. H., Dell'Acqua, M., Hibler, D. W., Pourmotabbed, T., & Gerlt, J. A. (1988) Biochemistry 27, 4127], suggest that lysine substitutions are not conservative in SNase and disrupt the conformation of the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Equilibrium denaturation of human growth hormone and its cysteine-modified forms.

The equilibrium denaturation of human growth hormone (hGH) derived from heterologous gene expression in Escherichia coli was studied. Denaturation was measured by ultraviolet absorbance, intrinsic fluorescence, far ultraviolet circular dichroism, and size exclusion chromatography. The denaturation transitions obtained from each method of detection were coincident, indicating a two-state denaturation mechanism. The denaturation transitions were independent of the concentration of protein. The Gibbs free energy of unfolding is 14.5 +/- 1 kcal/mol. Human growth hormone contains two disulfide bridges between residues 53-165 (large loop) and 182-189 (small loop). The small loop was selectively reduced and cysteines alkylated with iodoacetic acid or iodoacetamide. The tetra-S-carbamidomethylated and tetra-S-carboxymethylated derivatives were also prepared. All S-alkylated hGH forms were indistinguishable from the native conformations in the absence of denaturant by far ultraviolet circular dichroism. The circular dichroism-detected equilibrium denaturation of each derivative was determined and the Gibbs free energy of unfolding of the tetra-S-modified forms was 5.3 +/- 0.5 kcal/mol and of the di-S-alkylated derivatives was 11.2 +/- 0.8 kcal/mol. These results for hGH are different than previously obtained results for bovine, ovine, and rat growth hormones. Stable equilibrium intermediates have been identified for these non-human species of growth hormone. The stable intermediates observed in the denaturation of reduced, alkylated hGH or nonhunam growth hormones are similar and characterized as compact, helical, lacking native-like tertiary structure, and having a tendency to aggregate. The apparent absence of intermediates in the folding of oxidized hGH is due to the relative instability of intermediates compared with their native structures. The hGH conformation is at least 5 kcal/mol more stable than the growth hormones from other species. Reduction and alkylation of the disulfide bridges of hGH diminish the stability differences between the native and intermediate states, such that the denaturation behavior is similar to the nonhuman growth hormones with well-populated intermediates. Most proteins do not demonstrate equilibrium folding intermediates presumably because intermediates are only marginally stable in conditions that disrupt the native state. The folding results with hGH and alkylated hGH substantiate this.

Thermal Denaturation of Whey Proteins in Mixtures with Caseins Studied by Differential Scanning Calorimetry

Thermal unfolding of whey proteins and whey protein-casein mixtures in simulated milk ultrafiltrate was studied by differential scanning calorimetry. The results are reported as transition enthalpy, temperature of peak maximum, and van’t Hoff enthalpy related to the sharpness of transition. The results of earlier investigations were reviewed. It is suggested that a large part of reported whey protein unfolding data are affected by subsequent aggregation reactions.Pure caseins showed no endotherms on heating. In mixtures of whey proteins and individual caseins, β-casein showed no effect. Addition of 5% α-casein to 5% whey protein lowered the denaturation temperature of all whey proteins by 2 to 3°C. κ-Casein did not affect the denaturation behavior of bovine serum albumin and α-lactalbumin, but it lowered the denaturation temperature of β-lactoglobulin by 3°C. In the pH range from 6.4 to 7, where the whey protein casein interaction is known to change drastically in highly heated milk, no abrupt changes are seen in thermal unfolding of β-lactoglobulin, either alone or in mixture with κ-casein.

Thermodynamics of thermal and athermal denaturation of .gamma.-crystallins: changes in conformational stability upon glutathione reaction

The conformational stabilities of bovine lens gamma-crystallin fractions II, IIIA, IIIB, and IVA and those modified with glutathione were compared by studying the thermal and guanidine hydrochloride (Gdn-HCl) denaturation behavior. The conformational state was monitored by both far-UV CD and fluorescence measurements. All the gamma-crystallins studied showed a sigmoidal order-disorder transition with varied melting temperatures. The thermal denaturation of these proteins is reversible up to a temperature 3 or 4 degrees C above T 1/2; above this temperature, irreversible aggregation occurs. The validity of a two-state approximation of both thermal and Gdn-HCl denaturation was tested for all four crystallins, and the presence of one or more intermediates was evident in the unfolding of IVA. delta GDH2O values of these crystallins range from 4 to 9 kcal/mol. Upon glutathione treatment IVA showed the maximum decrease in T 1/2 by approximately 9 degrees C and in delta GDH2O value by 29%; the smallest decrease in T 1/2 was for IIIA by 2 degrees C and in delta GDH2O by 15%. We have demonstrated that the glutathione reaction can dramatically reduce the conformational stability of gamma-crystallins and, thus, that the thermodynamic quantities of the unreacted crystallins can be used to evaluate the stability of these proteins when modified during cataract formation.

Analysis of the thermal unfolding of porcine procarboxypeptidase A and its functional pieces by differential scnning calorimetry

Porcine pancreatic procarboxypeptidase A and its tryptic peptides, carboxypeptidase A and the activation segment, have been studied by high-sensitivity differential scanning calorimetry (DSC). The thermal denaturation of the zymogen and the active enzyme has been carried out at two pH values, 7.5 and 9.0, at different ionic strengths and at different scan rates. The endothermic transitions for these two proteins were always irreversible under all conditions investigated. The denaturation behaviour of both proteins seems to fit very well with the kinetic model for the DSC study of irreversible unfolding of proteins recently proposed by one of our groups. From this model, the activation energies obtained for the denaturation of the pro- and carboxypeptidase were 300 +/- 20 kJ mol-1 and 250 +/- 14 kJ mol-1 respectively. On the other hand, the isolated activation segment appears as a thermostable piece with a highly reversible thermal unfolding which follows a two-state process. The denaturation temperature observed for the isolated segment was always at least 15 K higher than those of the zymogen and the active enzyme.

Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction.

By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change delta Happ on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 +/- 0.2 degrees C and a delta Happ of 86.8 +/- 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 degrees C and 96 +/- 2 kcal/mol. The heat capacity change on denaturation delta Cp was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of delta Happ and delta Sapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated delta Cp for the mutant proteins and the d(delta Gapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.

Structural similarities among the high molecular weight protein fractions of oilseeds

Data on the physico-chemical properties of proteins from soybean, groundnut, sesame seed, sunflower seed, safflower seed, mustard seed, rapeseed and cotton seed are fairly extensive. An examination of the available data on high molecular weight proteins suggests that there are similarities in many of their properties. In this report the similarity in amino acid composition, size and shape, molecular weight, secondary structure, subunit composition, association-dissociation at high and low pH, stability towards denaturants, hydrolysis by enzymes and quaternary structure of the high molecular weight proteins is discussed. Based on these similarities a model has been proposed for the associationdissociation, denaturation and reassociation behaviour of the high molecular weight proteins of oilseeds.

Structure and stability of gamma-crystallins. Denaturation and proteolysis behavior.

The denaturation behavior of bovine lens gamma-crystallin fractions II, III, and IV and their susceptibility to proteolysis in vitro was compared to determine whether differences in their stability could play a role in cataract formation. Tertiary and secondary structure changes induced by increasing concentrations of urea, guanidine hydrochloride, and sodium dodecyl sulfate and by increasingly alkaline pH were followed by near-UV and far-UV circular dichroism, Trp fluorescence emission, and exposure of sulfhydryl groups. Major differences were found in the denaturation and proteolysis behavior of the three gamma-crystallin fractions. In general, the unfolding of gamma-II and gamma-III crystallins is rather gradual, suggesting the presence of intermediate unfolding states; in contrast, the order-disorder transition of gamma-IV crystallin is abrupt. The gamma-IV crystallin fraction is the most stable in urea and guanidine hydrochloride, but is most susceptible to nonspecific proteolysis and alkaline pH denaturation. Differences in denaturation and proteolysis behavior are attributed to the inherent differences in the tertiary structures of these crystallins.

Physical and chemical changes resulting from heat treatment of soya and soya alginate mixtures

The effect of water content and hydrocolloid inclusion on the denaturation behaviour and water binding properties of soya protein has been investigated mainly by using differential scanning calorimetry. The most interesting observation made was that when soya isolates containing high mannuronate alginates are heated there is a substantial increase in the amount of volatiles (probably water) formed. The dependence of this effect on water activity (a w ) suggests that it is the result of browning or condensation reactions. This effect is not seen to the same extent with other added hydrocolloids and may be the reason why alginate addition reduces the viscosity of the soya ‘melt’ during soya extrusion. The inclusion of high mannuronate alginate appears to raise the onset denaturation temperature of the 7S globulin and at low water contents reduces the heat change associated with the transition. It is possible that the former effect may be associated with the production of water during heating, which makes it difficult to define the water content at which denaturation is taking place. Measurements of freezable water and sorption isotherms suggest that alginate addition increases the water binding ability of soya isolate after denaturation.

Reoxidation of reduced bovine growth hormone from a stable secondary structure.

In order to determine solution conditions appropriate for reoxidizing reduced bovine growth hormone (bGH), we have examined the possibility of using a particular denaturant concentration to poise the secondary and tertiary structure of the reduced protein in a stable, nativelike state. It was envisioned that the structure of the reduced molecule would differ from that of the final oxidized molecule solely by the absence of disulfide bonds. Dilution of concentrated samples of reduced and unfolded protein from 6.0 M guanidine into 4.5 M urea followed by air oxidation indicated it was possible to induce refolding and reoxidation to an oxidized monomeric species in high yield (approximately 90%). The choice of solution conditions was based on comparison of urea equilibrium denaturation data for native oxidized protein to those for completely reduced protein and to protein in which sulfhydryl groups had been either partially or completely reduced and subjected to modification with iodoacetamide or methyl methanethiolsulfonate. The denaturation behavior of these species supports the existence of equilibrium folding intermediates for bovine growth hormone and demonstrates that chemical modification of the protein is capable of inducing differences in the denaturation behavior of these intermediates. The changes in the protein absorption spectrum and helix-related circular dichroism signal, along with direct titration of protein sulfhydryl groups, indicated that the refolding/reoxidation of bGH is a multistate process. The ordered nature of the kinetic changes in these probes during reoxidation indicates that disulfide formation is a sequential process, with little mispairing in 4.5 M urea, and that it proceeds through one or more obligatory kinetic folding events. The equilibrium denaturation behavior of the oxidized molecule and the various chemically modified forms, together with the reoxidation data, indicated that the protein maintains a high degree of secondary structure without intrachain disulfide bonds. The formation of these disulfide bonds is a discrete process which occurs after a framework of protein secondary structure is established.

Evidence that the conformational stability of ‘aged’ organophosphate-inhibited cholinesterase is altered

In order to determine whether a structural modification at the active center of cholinesterase may alter the conformational stability of the enzyme we compared the urea-induced unfolding of the tetrameric form of non-inhibited and irreversibly inhibited human plasma cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8). We studied enzyme inhibited by methanesulfonyl fluoride, diisopropylfluorophosphonate (DFP) and racemic soman. DFP- and soman-inhibited cholinesterases are converted spontaneously into non-reactivable forms called ‘aged’ enzymes through a process involving dealkylation of the bound organophosphate residue. The unfolding was followed by transverse urea-gradient polyacrylamide electrophoresis at various temperatures ranging from 0 to 60°C. Unfolding of cholinesterase appears to be a complex process. The denaturation patterns showed that partially unfolded states are thermodynamically unstable, but that several intermediates are involved; the lifetime of these depends on the temperature at which electrophoreses are carried out. Cholinesterase inhibited by methanesulfonyl fluoride behaved like the non-inhibited enzyme. On the other hand, small but significant differences in stability between non-inhibited and aged enzymes were observed. Whatever the temperature, the urea concentration at the mid-point of transition was always greater for aged enzyme than for the non-inhibited enzyme. In addition, aged enzymes showed more complex denaturation patterns at the lower temperatures (under 20°C). These findings suggest that the overall stability of aged-cholinesterases is slightly increased as compared with the stability of non-inhibited or methanesulfonyl fluoride-inhibited enzymes. The denaturation pattern obtained at 0°C for soman-inhibited cholinesterase under non-aging conditions (inhibition at 0°C, pH 10.7) was similar to that of non-inhibited enzyme at this temperature, although splitting in two of the denaturation curve over the transition zone reflects the heterogeneity of soman-inhibited enzyme. The slight difference in denaturation behavior between these species may be due to stereoisomerism in soman. The differences in electrophoretic behavior and apparent stability observed between non-inhibited and aged enzymes were interpreted as the result of a conformational change induced by the dealkylation reaction of enzyme-inhibitor conjugates.

Isotopic effect on the kinetic of thermal denaturation of ceruloplasmin.

The kinetics of thermal denaturation of ceruloplasmin in water and in water with different percentage of D2O in the temperature range 25-85 degrees C, following the optical density change of the 610 nm charge transfer band of the protein has been investigated. A temperature Tt approximately equal to 65 degrees C above which an irreversible denaturation process of the protein active site takes place has been found. The kinetics of the denaturation process of the protein are fitted by two first order reactions, which have been assigned to a different thermal denaturation behaviour of the two Cu2+ type I sites existing in the protein. Addition of D2O to the protein solution affects the two kinetics in a different way, i.e. the rate of one of them is increased whilst that of the other is decreased. The different effect of D2O on the kinetics of disruption of the two copper sites is discussed in terms of different location and degree of hydrophobicity of the two Cu2+ type I sites.

Preliminary Characterization of an Antibiotic Produced by Xanthomonas albilineans Which Inhibits DNA Synthesis in Escherichia coli

Chlorosis-inducing isolates of Xanthomonas albilineans, the sugarcane leaf scald pathogen, produced a mixture of antibacterial compounds in culture. The antibiotic mixture, which eluted as a single strongly retarded peak from Sephadex LH-20 in methanol, was bactericidal to Escherichia coli. Inhibition of E. coli was not reversed by added nutrients, and affected cells were not lysed but many accumulated polyphosphate granules. The major antibacterial component, isolated in crystalline form after HPLC, is given the trivial name albicidin. Near the minimum inhibitory concentration, albicidin caused a complete block to DNA synthesis, followed by partial inhibition of RNA and protein synthesis, as assessed by incorporation of radioactive precursors. Spontaneous antibiotic-resistant mutants of E. coli showed no cross-resistance between albicidin and inhibitors of either subunit of DNA gyrase. Mixing albicidin with purified DNA from E. coli did not alter the thermal denaturation behaviour of the DNA, or the absorption spectrum of the antibiotic. PolA+ and PolA - strains of E. coli were equally sensitive to albicidin, indicating that the antibiotic does not bind to or modify DNA. Selective inhibition of DNA synthesis without evidence of DNA binding suggests a specific interaction of albicidin with an essential replication protein.

Denaturation behavior of antithrombin in guanidinium chloride. Irreversibility of unfolding caused by aggregation.

The structural stability of the protease inhibitor antithrombin from bovine plasma was examined as a function of the concentration of guanidinium chloride (GdmCl). A biphasic unfolding curve at pH 7.4, with midpoints for the two phases at 0.8 and 2.8 M GdmCl, was measured by far-ultraviolet circular dichroism. Spectroscopic and hydrodynamic analyses suggest that the intermediate state which exists at 1.5 M GdmCl involves a partial unfolding of the antithrombin molecule that exposes regions of the polypeptide chain through which slow, intermolecular association subsequently takes place. The partially unfolded molecule can be reversed to its fully functional state only before the aggregation occurs. Upon return of the aggregated state to dilute buffer, the partially unfolded antithrombin remains aggregated and does not regain the spectroscopic properties, thrombin-inhibitory activity, or heparin affinity of the native inhibitor. This behavior indicates that the loss of the functional properties of the proteins is caused by the macromolecular association. Comparative experiments gave similar results for the human inhibitor. Analyses of bovine antithrombin in 6 M GdmCl indicated that the second transition reflects the total unfolding of the protein to a disulfide-cross-linked random coil. This transition is spectroscopically reversible; however, on further reversal to dilute buffer, the molecules apparently are trapped in the partially unfolded, aggregated, intermediate state. The results are consistent with the existence of two separate domains in antithrombin which unfold at different concentrations of GdmCl but do not support the contention that the thrombin-binding and heparin-binding regions of the protein are located in different domains [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053].

Conformation and processing of cathepsin D.

Cathepsin D occurs in two forms, a single polypeptide chain (Mr 44 000) and a non-covalent complex of two peptides of Mr 14 000 and 30 000 that is derived by proteolytic processing of the 44 000 polypeptide. The two forms from bovine spleen are closely similar in secondary structure content, in aromatic amino acid environment and in the two step denaturation behaviour. Enzyme activity is lost irreversibly on denaturation but conformation can be partially regained. The two separated chains will only refold partially and this is related to their positions in the overall structure of cathepsin D. It is suggested that the processing step is related to protein turnover.

Conformation, structure and activation of bovine cathepsin D. Unfolding and refolding studies.

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the other a non-covalent complex of two peptides of Mr 14 000 and 30 000. These correspond to the N-terminal and C-terminal regions of the single chain from which they originate. It has been shown that the two forms of the enzyme are closely similar in secondary-structure content, in aromatic amino acid environment and in denaturation behaviour. The two-chain enzyme has half the specific activity of the single-chain form. The denaturation and renaturation of the single-chain cathepsin D has now been studied by c.d., fluorescence and enzyme activity. Activity is lost irreversibly on unfolding, but the loss of backbone ellipticity and of folded aromatic environment is 75% reversible. The enzyme unfolds in two main stages, and the kinetics of these transitions indicate the existence of at least two intermediate forms between the native and the fully unfolded states. A further form of the enzyme exists in 0.5 M-guanidinium chloride. It is characterized by having an activity 40% greater than that of the native state. This increase is not reversed on removing the denaturant. The similarities between cathepsin D and pepsin are discussed.

The denaturation of orosomucoid

Highly purified human orosomucoid exhibits apparent multistate behavior on thermal denaturation, with Δ H(cal) for the transition being 119 kcal/mol at pH 7.4. Asialoorosomucoid denatured similarly. A domain structure could not be demonstrated for orosomucoid with cyanogen bromide fragments, although some reannealing of these did occur. It is suggested that the source of the apparent multistate behavior may lie in the existence of polypeptide variants, known to be present in orosomucoid. Although some polymerization occurs on heating, the unpolymerized material shows reversible thermal denaturation behavior. The presence of low concentrations of ethanol induces a significant endotherm which moves to lower temperatures with increasing ethanol concentration. This endotherm was irreversible in the continued presence of ethanol.

A differential scanning calorimetric study of the thermal denaturation of bovine β-lactoglobulin Thermal behaviour at temperatures up to 100°C

The thermal behaviour of β-lactoglobulin in aqueous solutions was followed by differential scanning calorimetry in the temperature range from 40 to 100°C. From the results the following information is obtained. 1. 1. The DSC thermograms show a large transition peak in the temperature range from 60 to 90°C, which reflects the denaturation (unfolding) of β-lactoglobulin. 2. 2. From the peak surface an apparent denaturation enthalpy ΔH = 230 ± 15 kJ/mol is calculated at pH 6.5. 3. 3. The temperature of maximum deflection of the DSC curve is dependent on the heating rate. Extrapolation to zero heating rate results in a denaturation temperature of 70.4 ± 0.5° C at pH 6.7. 4. 4. A kinetic analysis of the DSC curves shows that the denaturation of β-lactoglobulin is of the first order at temperatures between 65 to 72°C. The apparent activation energy amounts to 343 kJ/mol, calculated according to the method of Kissinger. After comparison of the results with data from the literature, it was concluded that 70°C is a critical temperature for the denaturation of β-lactoglobulin. Above 70°C the denaturation behaviour is changed, probably because of the starting of the aggregation process. This change is indicated by the transition temperature of the DSC curve at the corresponding heating rate.

Stability differences between high coagulant (alpha) and noncoagulant (gamma) human thrombins. Denaturation.

Human alpha-thrombin, the two (covalently linked)-chain, highly coagulant blood-clotting enzyme was compared with its noncoagulant, yet estero/amidolytically active derivative, gamma-thrombin, a three (noncovalently associated)-domain enzyme which results from two proteolytic cleavages of the coagulant a form. Studies of their denaturation behavior by Tos-Arg-OMe esterase activity, by intrinsic fluorescence, by fluorescence of active serine-directed dansyl labels, and by monitoring the ESR of a fluorosulfonylphenyl spin-labeled inhibitor clearly demonstrated the reduced stability of the noncovalently associated gamma-thrombin form. At pH 6.5, 0.75 M NaCl, gamma-thrombin unfolds in approximately 2. 1 M urea while the more stable a form denatures at approximately 4 M urea. By monitoring active serine probes (spin label or fluorescent labels), these transitions were slightly lower, 1.0 +/- 0.1 and 2.8 +/- 0.2 M urea for spin-labeled gamma- and alpha-thrombins, respectively. Similar behavior was found for the same spin-labeled derivatives in guanidine HCl with unfolding transitions of 0.4 M and 1.0 M for spin-labeled gamma- and alpha-thrombin, respectively. These differences in structural stabilization serve as a good physical diagnostic for the two thrombin species.

Effect of sodium dodecyl sulfate, acid, alkali, urea and guanidine hydrochloride on the circular dichroism of alpha-globulin of Sesamum indicum L.

The circular dichrotic spectra of alpha-globulin have been obtained under various solution conditions of sodium dodecyl sulfate, acid, alkali, urea and guanidine hydrochloride. The protein in phosphate buffer pH 7.4, 0.2 M has about 25% beta-structure and 5% alpha-helix, the rest being aperiodic or irregular structure. Sodium dodecyl sulfate induced more alpha-helical structure in the protein. The protein had nearly 20% alpha-helix at 1 X 10(-2) M SDS. At extreme acid or alkaline pH, the protein had no alpha-helix with beta-structure decreasing with further extremes of pH. The protein is represented by 100% aperiodic structure in 6.6 M urea and in 6.0 M guanidine hydrochloride solutions. The above results are discussed in view of some of the earlier results with regard to the association-dissociation and denaturation behavior of alpha-globulin under various solution conditions.