pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1

To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.     

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.     

Comparison of inactivation and unfolding of methanol dehydrogenase during denaturation in guanidine hydrochloride and urea

The activity and the conformational changes of methanol dehydrogenase (MDH), a quinoprotein containing pyrrolo-quinoline quinone as its prosthetic group, have been studied during denaturation in guanidine hydrochloride (GdnHCl) and urea. The unfolding of MDH was followed using the steady-state and time resolved fluorescence methods. Increasing the denaturant concentration in the denatured system significantly enhanced the inactivation and unfolding of MDH. The enzyme was completely inactivated at 1 M GdnHCl or 6 M urea. The fluorescence emission maximum of the native enzyme was at 332 nm. With increasing denaturant concentrations, the fluorescence emission maximum red-shifted in magnitude to a maximum value (355 nm) at 5 M GdnHCl or 8 M urea. Comparison of inactivation and conformational changes during denaturation showed that in general accord with the suggestion made previously by Tsou, the active sites of MDH are situated in a region more flexible than the molecule as a whole.     

Mechanism of Unfolding of Goat Lung Cystatin During Urea and Guanidine Hydrochloride Induced Denaturation

Cystatins essentially regulate lysosomal cysteine protease besides affecting several physiological processes. In the present study, denaturation of a high molecular weight cystatin (Mr 66.4 kDa) purified from goat lung (GLC-I) has been studied by monitoring its inhibitory activity, intrinsic fluorescence, circular dichroism (CD), and binding of ANS. It was found that increasing concentration of GdnHCl significantly enhances the inactivation and unfolding of the purified inhibitor (GLC-I) with complete loss of inhibitory activity at 4 M GdnHCl. Denaturation of GLC-I in the presence of GdnHCl is accompanied by red shift (15 nm) of the emission maximum as shown by intrinsic fluorescence. The inhibitory activity of GLC-I was increased by 1.5 fold at 2 M urea; however, it decreased with further increased of the urea concentration. Intrinsic fluorescence studies of GLC-I in the presence of 0–3 M urea shows blue shift of 5 nm, suggesting stabilization of the inhibitor followed by 5 nm red shift at higher concentration. ANS binding studies in the presence of urea indicate significant changes in the tertiary structure of the inhibitor. Thus, our result shows denaturation profile of GLC-I following simple two state transitions in the presence of GdnHCl while it proceeds through an intermediate state in the presence of urea.     

Hydrogen exchange kinetics of RNase A and the urea:TMAO paradigm

A key paradigm in the biology of adaptation holds that urea affects protein function by increasing the fluctuations of the native state, while trimethylamine N-oxide (TMAO) affects function in the opposite direction by decreasing the normal fluctuations of the native ensemble. Using urea and TMAO separately and together, hydrogen exchange (HX) studies on RNase A at pH* 6.35 were used to investigate the basic tenets of the urea:TMAO paradigm. TMAO (1 M) alone decreases HX rate constants of a select number of sites exchanging from the native ensemble, and low urea alone increases the rate constants of some of the same sites. Addition of TMAO to urea solutions containing RNase A also suppresses HX rate constants. The data show that urea and TMAO independently or in combination affect the dynamics of the native ensemble in opposing ways. The results provide evidence in support of the counteraction aspect of the urea:TMAO paradigm linking structural dynamics with protein function in urea-rich organs and organisms. RNase A is so resistant to urea denaturation at pH* 6.35 that even in the presence of 4.8 M urea, the native ensemble accounts for >99.5% of the protein. An essential test, devised to determine the HX mechanism of exchangeable protons, shows that over the 0-4.8 M urea concentration range nearly 80% of all observed sites convert from EX2 to EX1. The slow exchange sites are all EX1; they do not exhibit global exchange even at urea concentrations (5.8 M) well into the denaturation transition zone, and their energetically distinct activated complexes leading to exchange gives evidence of residual structure. Under these experimental conditions, the use of DeltaG(HX) as a basis for HX analysis of RNase A urea denaturation is invalid.     

Bromophenol blue binding as a probe to study urea and guanidine hydrochloride denaturation of bovine serum albumin

Urea and guanidine hydrochloride (GdnHCl) denaturation of bovine serum albumin (BSA) were investigated using bromophenol blue (BPB) binding as a probe. Addition of BPB to BSA produced an absorption difference spectrum in the wavelength range, 525-675 nm with a minimum at 587 nm and a maximum at 619 nm. The magnitude of absorption difference (DeltaAbs.) at 619 nm decreased on increasing urea/GdnHCl concentration and followed the denaturation curve. The denaturation was found to be a two-state, single-step transition. The transitions started at 1.75 and 0.875 M and completed at 6.5 and 3.25 M with the mid point occurring around 4.0 and 1.5 M urea and GdnHCl concentrations, respectively. The value of free energy of stabilization, DeltaGDH2O as determined from urea and GdnHCl denaturation curves was found to be 4041 and 4602 cal/mol, respectively. Taken together, these results suggest that BPB binding can be used as a probe to study urea and GdnHCl denaturation of BSA.     

States of tryptophyl residues and stability of recombinant human matrix metalloproteinase 7 (matrilysin) as examined by fluorescence

States of tryptophyl residues and stability of human matrilysin were studied. The activation energy for the thermal inactivation of matrilysin was determined to be 237 kJ/mol, and 50% of the activity was lost upon incubation at 69 degrees C for 10 min. The activity was increased by adding NaCl, and was doubled with 3 M NaCl. Denaturation of matrilysin by guanidine hydrochloride (GdnHCl) and urea was monitored by fluorescence change of tryptophyl residues. Half of the change was observed at 2.2-2.7 M GdnHCl, whereas no change was observed even with 8 M urea. Half of the inactivation was induced at 0.8 M GndHCl and at 2 M urea. The presence of an inactive intermediate with the same fluorescence spectrum as the native enzyme was suggested in the denaturation. Matrilysin contains four tryptophyls, and their states were examined by fluorescence-quenching with iodide and cesium ions and acrylamide. No tryptophyls in the native enzyme were accessible to I(-) and Cs(+), and 2.4 residues were accessible to acrylamide. Based on the crystallographic study, Trp154 is water-accessible, but it should be in a crevice not to contact with I(-) and Cs(+). All tryptophyls in the GdnHCl-denatured enzyme were exposed to the quenchers, while a considerable part was inaccessible in the urea-denatured one.     

Structural stability of the PsbQ protein of higher plant photosystem II

We have characterized the stability and folding behavior of the isolated extrinsic PsbQ protein of photosystem II (PSII) from a higher plant, Spinacia oleracea, using intrinsic protein fluorescence emission and near- and far-UV circular dichroism (CD) spectroscopy in combination with differential scanning calorimetry (DSC). Experimental results reveal that both chemical denaturation using guanidine hydrochloride (GdnHCl) and thermal unfolding of PsbQ proceed as a two-state reversible process. The denaturation free-energy changes (DeltaG(D)) at 20 degrees C extrapolated from GdnHCl (4.0 +/- 0.6 kcal mol(-1)) or thermal unfolding (4.4 +/- 0.8 kcal mol(-1)) are very close. Moreover, the far-UV CD spectra of the denatured PsbQ registered at 90 degrees C in the absence and presence of 6.0 M GdnHCl superimpose, leading us to conclude that both denatured states of PsbQ are structurally and energetically similar. The thermal unfolding of PsbQ has been also characterized by CD and DSC over a wide pH range. The stability of PsbQ is at its maximum at pH comprised between 5 and 8, being wider than the optimal pH for oxygen evolution in the lumen of thylakoid membranes. In addition, no significant structural changes were detected in PsbQ between 50 and 55 degrees C in the pH range of 3-8, suggesting that PsbQ behaves as a soluble and stable particle in the lumen when it detaches from PSII under physiological stress conditions such as high temperature (45-50 degrees C) or low pH (<5.0). Sedimentation experiments showed that, in solution at 20 degrees C, the PsbQ protein is a monomer with an elongated shape.     

A stability transition at mildly acidic pH in the alpha-hemolysin (alpha-toxin) from Staphylococcus aureus.

The effects of mildly acidic conditions on the free energy of unfolding (DeltaG(u)(buff)) of the pore-forming alpha-hemolysin (alphaHL) from Staphylococcus aureus were assessed between pH 5.0 and 7.5 by measuring intrinsic tryptophan fluorescence, circular dichroism and elution time in size exclusion chromatography during urea denaturation. Decreasing the pH from 7.0 to 5.0 reduced the calculated DeltaG(u)(buff) from 8.9 to 4.2 kcal mol(-1), which correlates with an increased rate of pore formation previously observed over the same pH range. It is proposed that the lowered surface pH of biological membranes reduces the stability of alphaHL thereby modulating the rate of pore formation.     

The peculiar nature of the guanidine hydrochloride-induced two-state denaturation of staphylococcal nuclease: a calorimetric study.

This work determines the ratio of DeltaH(vH) /DeltaH(cal) for staphylococcal nuclease (SN) denaturation in guanidine hydrochloride (GdnHCl) to test whether GdnHCl-induced denaturation is two-state. Heats of mixing of SN as a function of [GdnHCl] were determined at pH 7.0 and 25 degrees C. The resulting plot of DeltaH(mix) vs [GdnHCl] exhibits a sigmoid shaped curve with linear pre- and post-denaturational base lines. Extending the pre- and post-denaturational lines to zero [GdnHCl] gives a calorimetric DeltaH (DeltaH(cal)) of 24.1 +/- 1.0 kcal/mol, for SN denaturation in the limit of zero GdnHCl concentration. Guanidine hydrochloride-induced denaturation Gibbs energy changes in the limit of zero denaturant concentration (DeltaG degrees (N)(-)(D)) at pH 7. 0 were determined for SN from fluorescence measurements at fixed temperatures over the range from 15 to 35 degrees C. Analysis of the resulting temperature-dependent DeltaG degrees (N)(-)(D) data defines a van't Hoff denaturation enthalpy change (DeltaH(vH)) of 26. 4 +/- 2.8 kcal/mol. The model-dependent van't Hoff DeltaH(vH) divided by the model-independent DeltaH(cal) gives a ratio of 1.1 +/- 0.1 for DeltaH(vH)/DeltaH(cal), a result that rules out the presence of thermodynamically important intermediate states in the GdnHCl-induced denaturation of SN. The likelihood that GdnHCl-induced SN denaturation involves a special type of two-state denaturation, known as a variable two-state process, is discussed in terms of the thermodynamic implications of the process.     

Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions.

The objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled-coil analogs were designed, where the number of intrachain and interchain electrostatic attractions (A) were systematically changed to repulsions (R): 20A, 15A5R, 10A10R, and 20R. The GdnHCl denaturation data showed that the 4 coiled-coil analogs, which had electrostatic interactions ranging from 20 attractions to 20 repulsions, had very similar [GdnHCl]1/2 values (average of congruent to 3.5 M) and, as well, their delta delta Gu values were very close to 0 (0.2 kcal/mol). In contrast, urea denaturation showed that the [urea]1/2 values proportionately decreased with the stepwise change from 20 electrostatic attractions to 20 repulsions (20A, 7.4 M; 15A5R, 5.4 M; 10A10R, 3.2 M; and 20R, 1.4 M), and the delta delta Gu values correspondingly increased with the increasing differences in electrostatic interactions (20A-15A5R, 1.5 kcal/mol; 20A-10A10R, 3.7 kcal/mol; and 20A-20R, 5.8 kcal/mol). These results indicate that the ionic nature of GdnHCl masks electrostatic interactions in these model proteins, a phenomenon that was absent when the unchanged urea was used. Thus, GdnHCl and urea denaturations may give vastly different estimates of protein stability, depending on how important electrostatic interactions are to the protein.     

Trimethylamine N-oxide counteracts the denaturing effects of urea or GdnHCl on protein denatured state

To understand trimethylamine N-oxide (TMAO) attenuation of the denaturating effects of urea or guanidine hydrochloride (GdnHCl), we have determined the apparent transfer free energies (DeltaG(tr)(')) of cyclic dipeptides (CDs) from water to TMAO, urea or GdnHCl, and also the blends of TMAO and denaturants (urea or GdnHCl) at a 1:2 ratio as well as various denaturant concentrations in the presence of 1M TMAO, through the solubility measurements, at 25 degrees C. The CDs investigated in the present study included cyclo(Gly-Gly), cyclo(Ala-Ala) and cyclo(Val-Val). The observed DeltaG(tr)(') values indicate that TMAO can stabilize the CDs while urea or GdnHCl can destabilize the CDs. Furthermore, the DeltaG(tr)(') values of the blends of TMAO with urea or GdnHCl revealed that TMAO strongly counteracted the denaturating effects of urea on CDs in all instances, however, TMAO partially counteracted the perturbing effects of GdnHCl on CDs. TMAO counteraction ability of the deleterious effects of denaturants depended on the denaturant-CDs pair. The experimental results were further used to estimate the transfer free energies (Deltag(tr)(')) of the various functional group contributions from water to TMAO, urea or GdnHCl individually and to the combinations of TMAO and the denaturants in various ratios.     

Denaturation induced aggregation in α‐crystallin: differential action of chaotropes

α‐Crystallin is a member of small heat shock proteins and is believed to play an exceptional role in the stability of eye lens proteins. The disruption or denaturation of the protein arrangement or solubility of the crystallin proteins can lead to vision problems including cataract. In the present study, we have examined the effect of chemical denaturants urea and guanidine hydrochloride (GdnHCl) on α‐crystallin aggregation, with special emphasis on protein conformational changes, unfolding, and amyloid fibril formation. GdnHCl (4 M) induced a 16 nm red shift in the intrinsic fluorescence of α‐crystallin, compared with 4 nm shift by 8 M urea suggesting a major change in α‐crystallin structure. Circular dichroism analysis showed marked increase in the ellipticity of α‐crystallin at 216 nm, suggesting gain in β‐sheet structure in the presence of GdnHCl (0.5–1 M) followed by unfolding at higher concentration (2–6 M). However, only minor changes in the secondary structure of α‐crystallin were observed in the presence of urea. Moreover, 8‐anilinonaphthalene‐1‐sulfonic acid fluorescence measurement in the presence of GdnHCl and urea showed changes in the hydrophobicity of α‐crystallin. Amyloid studies using thioflavin T fluorescence and congo red absorbance showed that GdnHCl induced amyloid formation in α‐crystallin, whereas urea induced aggregation in this protein. Electron microscopy studies further confirmed amyloid formation of α‐crystallin in the presence of GdnHCl, whereas only aggregate‐like structures were observed in α‐crystallin treated with urea. Our results suggest that α‐crystallin is susceptible to unfolding in the presence of chaotropic agents like urea and GdnHCl. The destabilized protein has increased likelihood to fibrillate. Copyright © 2016 John Wiley & Sons, Ltd.     

Hydrogen-exchange stabilities of RNase T1 and variants with buried and solvent-exposed Ala --> Gly mutations in the helix

Hydrogen-exchange rates were measured for RNase T1 and three variants with Ala --> Gly substitutions at a solvent-exposed (residue 21) and a buried (residue 23) position in the helix: A21G, G23A, and A21G + G23A. These results were used to measure the stabilities of the proteins. The hydrogen-exchange stabilities (DeltaG(HX)) for the most stable residues in each variant agree with the equilibrium conformational stability measured by urea denaturation (DeltaG(U)), if the effects of D(2)O and proline isomerization are included [Huyghues-Despointes, B. M. P., Scholtz, J. M., and Pace, C. N. (1999) Nat. Struct. Biol. 6, 210-212]. These residues also show similar changes in DeltaG(HX) upon Ala --> Gly mutations (DeltaDeltaG(HX)) as compared to equilibrium measurements (DeltaDeltaG(U)), indicating that the most stable residues are exchanging from the globally unfolded ensemble. Alanine is stabilizing compared to glycine by 1 kcal/mol at a solvent-exposed site 21 as seen by other methods for the RNase T1 protein and peptide helix [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 3833-2837], while it is destabilizing at the buried site 23 by the same amount. For the A21G variant, only local NMR chemical shift perturbations are observed compared to RNase T1. For the G23A variant, large chemical shift changes are seen throughout the sequence, although X-ray crystal structures of the variant and RNase T1 are nearly superimposable. Ala --> Gly mutations in the helix of RNase T1 at both helical positions alter the native-state hydrogen-exchange stabilities of residues throughout the sequence.     

Autophosphorylation dependent destabilization of the insulin receptor kinase domain: tryptophan-1175 reports changes in the catalytic cleft

Protein kinases are regulated by conformational or chemical changes which facilitate access of substrates to the active site and promote correct orientations of catalytically essential residues and water molecules. The switch between basal and activated states of the insulin receptor's kinase domain (IRKD) results from autophosphorylation. We investigated the effects of IRKD autophosphorylation on the conformational stability by guanidine hydrochloride (GdnHCl) dependent denaturation and by iodide quenching of intrinsic fluorescence. Tryptophan residues of the recombinant soluble IRKD (residues R953-S1355) were excited at a lambdaex of 295 nm, and emission spectra were analyzed for centroid (a characteristic of average polarity of the indole rings' environments) and integrated fluorescence intensity over the lambdaem range of 310-420 nm. Denaturation profiles of both apo- and phospho-IRKD forms are complex with at least three distinct unfolding transitions. The first and last transitions were reversible and cooperative and had midpoints at 0.4 or 0.7 M GdnHCl and 2.4 or 2.7 M GdnHCl, respectively; transitions of phospho-IRKD occurred at lower GdnHCl concentrations. Calculations of free energy of unfolding suggested a loss of approximately 2.3 kcal/mol of stabilization for the first transition and approximately 1.5 kcal/mol for the third transition. Circular dichroism showed subtle changes in secondary structure over the first transition and global unfolding over the last transition. The first transition reports changes primarily in the local environment of W1175, which is near the catalytic loop and is conserved among protein tyrosine kinases. W1175 is also the dominant fluorophore of the native emission spectrum. Iodide quenching of W1175 was virtually undetectable in the apo-IRKD but significant in the phospho-IRKD, suggesting that W1175 exposure to small solutes is strongly dependent on the conformation of the activation loop. These studies indicate that autophosphorylation, while exposing the catalytic center, also produces a conformer less stable than the apoenzyme.     

pH dependence of the stability of barstar to chemical and thermal denaturation.

Equilibrium unfolding of barstar with guanidine hydrochloride (GdnHCl) and urea as denaturants as well as thermal unfolding have been carried out as a function of pH using fluorescence, far-UV and near-UV CD, and absorbance as probes. Both GdnHCl-induced and urea-induced denaturation studies at pH 7 show that barstar unfolds through a two-state F<->U mechanism and yields identical values for delta GU, the free energy difference between the fully folded (F) and unfolded (U) forms, of 5.0 +/- 0.5 kcal.mol-1 at 25 degrees C. Thermal denaturation of barstar also follows a two-state F<->U unfolding transition at pH 7, and the value of delta GU at 25 degrees C is similar to that obtained from chemical denaturation. The pH dependence of denaturation by GdnHCl is complex. The Cm value (midpoint of the unfolding transition) has been used as an index for stability in the pH range 2-10, because barstar does not unfold through a two-state transition on denaturation by GdnHCl at all pH values studied. Stability is maximum at pH 2-3, where barstar exists in a molten globule-like form that forms a large soluble oligomer. The stability decreases with an increase in pH to 5, the isoelectric pH of the protein. Above pH 5, the stability increases as the pH is raised to 7. Above pH 8, it again decreases as the pH is raised to 10. The decrease in stability from pH 7 to 5 in wild-type (wt) barstar, which is shown to be characterized by an apparent pKa of 6.2 +/- 0.2, is not observed in H17Q, a His 17-->Gln 17 mutant form of barstar. This decrease in stability has therefore been correlated with the protonation of His 17 in barstar. The decrease in stability beyond pH 8 in wt barstar, which is characterized by an apparent pKa of 9.2 +/- 0.2, is not detected in BSCCAA, the Cys 40 Cys 82-->Ala 40 Ala 82 double mutant form of barstar. Thus, this decrease in stability has been correlated with the deprotonation of at least one of the two cysteines present in wt barstar. The increase in stability from pH 5 to 3 is characterized by an apparent pKa of 4.6 +/- 0.2 for wt barstar and BSCCAA, which is similar to the apparent pKa that characterizes the structural transition leading to the formation of the A form. The use of Cm as an index of stability has been supported by thermal denaturation studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Apparent pKa shifts of titratable residues at high denaturant concentration and the impact on protein stability

Urea and guanidine-hydrochloride (GdnHCl) are frequently used for protein denaturation in order to determine the Gibbs free energy of folding and kinetic folding/unfolding parameters. Constant pH value is applied in the folding/unfolding experiments at different denaturant concentrations and steady protonation state of titratable groups is assumed in the folded and unfolded protein, respectively. The apparent side-chain pKa values of Asp, Glu, His and Lys in the absence and presence of 6 M urea and GdnHCl, respectively, have been determined by 1H-NMR. pKa values of all four residues are up-shifted by 0.3-0.5 pH units in presence of 6 M urea by comparison with pKa values of the residues dissolved in water. In the presence of 6 M GdnHCl, pKa values are down-shifted by 0.2-0.3 pH units in the case of acidic and up-shifted by 0.3-0.5 pH units in the case of basic residues. Shifted pKa values in the presence of denaturant may have a pronounced effect on the outcome of the protein stability obtained from denaturant unfolding experiments.     

pH dependence of stability of staphylococcal nuclease: evidence of substantial electrostatic interactions in the denatured state

The pH dependence of stability of staphylococcal nuclease was studied with two independent equilibrium thermodynamic approaches. First, by measurement of stability in the pH range 9 to 3.5 by fluorescence-monitored denaturation with urea (Delta), GdnHCl (Delta), and heat (Delta). Second, by numerical integration of H(+) titration curves (Delta) measured potentiometrically under native (100 mM KCl) and unfolding (6.0 M GdnHCl) conditions. The pH dependence of stability described by Delta, Delta, and Delta was comparable but significantly different from the one described by Delta. The decrease in Delta between pH 9 and pH 4 was 4 kcal/mol greater than the decrease in Delta, Delta, and Delta in the same pH range. In 6 M GdnHCl, all the ionizable groups titrated with the pK(a) values of model compounds. Therefore, Delta represents the free energy difference between the native state (N) and an ensemble of unstructured, or expanded, and highly screened conformations. In contrast, the shallower pH dependence of stability described by Delta and by Delta between pH 9 and 5 was consistent with the titration of histidines with depressed, nativelike pK(a) values in the denatured state (D). These depressed pK(a) values likely reflect long-range electrostatic interactions with the other 29 basic groups and are a consequence of the compact character of the D state. The steep change in Delta and Delta at pH < 5 suggests that near pH 5 the structural and thermodynamic character of the D state shifts toward a state in which acidic residues titrate with normal pK(a) values, presumably because the electrostatic interactions with basic residues are lost, maybe as a consequence of an expansion.     

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.