Thermodynamic analysis of denaturant-induced unfolding of HodC69S protein supports a three-state mechanism

Thermodynamic stability parameters and the equilibrium unfolding mechanism of His 6HodC69S, a mutant of 1 H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) having a Cys to Ser exchange at position 69 and an N-terminal hexahistidine tag (His 6HodC69S), have been derived from isothermal unfolding studies using guanidine hydrochloride (GdnHCl) or urea as denaturants. The conformational changes were monitored by following changes in circular dichroism (CD), fluorescence, and dynamic light scattering (DLS), and the resulting transition curves were analyzed on the basis of a sequential three-state model N = I = D. The structural changes have been correlated to catalytic activity, and the contribution to stability of the disulfide bond between residues C37 and C184 in the native protein has been established. A prominent result of the present study is the finding that, independent of the method used for denaturing the protein, the unfolding mechanism always comprises three states which can be characterized by, within error limits, identical sets of thermodynamic parameters. Apparent deviations from three-state unfolding can be rationalized by the inability of a spectroscopic probe to discriminate clearly between native, intermediate, and unfolded ensembles. This was the case for the CD-monitored urea unfolding curve.     

Revealing a Concealed Intermediate that Forms after the Rate-limiting Step of Refolding of the SH3 Domain of PI3 Kinase

Kinetic and equilibrium studies of the folding and unfolding of the SH3 domain of the PI3 kinase, have been used to identify a folding intermediate that forms after the rate-limiting step on the folding pathway. Folding and unfolding, in urea as well as in guanidine hydrochloride (GdnHCl), were studied by monitoring changes in the intrinsic fluorescence or in the far-UV circular dichroism (CD) of the protein. The two probes yield non-coincident equilibrium transitions for unfolding in urea, indicating that an intermediate, I, exists in equilibrium with native (N) and unfolded (U) protein, during unfolding. Hence, the equilibrium unfolding data were analyzed according to a three-state N ↔ I ↔ U mechanism. An intermediate is observed also in kinetic unfolding studies, and its presence leads to the unfolding reaction in urea as well as in GdnHCl, occurring in two steps. The fast step is complete within the initial 11 ms of unfolding and manifests itself in a burst phase change in fluorescence. At high concentrations of GdnHCl, the entire change in fluorescence during unfolding occurs during the 11 ms burst phase. CD measurements indicate, however, that I retains N-like secondary structure. An analysis of the kinetic and thermodynamic data, according to a minimal three-state N ↔ I ↔ U mechanism, positions I after the rate-limiting transition state, TS1, of folding, on the reaction coordinate of folding in GdnHCl. Hence, I is not revealed when folding is commenced from U, regardless of the nature of the probe used to follow the folding reaction. Interrupted unfolding experiments, in which the protein is unfolded transiently in GdnHCl for various lengths of time before being refolded, showed that I refolds to N much faster than does U, confirms the analysis of the direct folding and unfolding experiments, that I is formed after the rate-limiting step of refolding in GdnHCl.     

Evidence of stable monomeric species in the unfolding of Cu,Zn superoxide dismutase from Photobacterium leiognathi.

The equilibrium unfolding process of Photobacterium leiognathi Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism (CD) and fluorescence spectroscopy, upon increasing the guanidinium hydrochloride concentration. The study has been undertaken for both the holo- and the copper-free derivative to work out the role of copper in protein stability. In both cases the unfolding was reversible. The denaturation curve derived from CD and fluorescence spectroscopy was not coincident, suggesting that the denaturation process occurs through a three-state model with formation of an intermediate monomeric species. The occurrence of an intermediate species has been unambiguously demonstrated following CD and steady-state fluorescence spectra of the enzyme at various concentrations in presence of a fixed amounts of guanidinium hydrochloride.     

Identification of an active dimeric intermediate populated during the unfolding process of the cambialistic superoxide dismutase from Streptococcus mutans

Superoxide dismutases are enzymes that protect biological systems against oxidative damage caused by superoxide radicals. In this paper, a detailed characterization is presented on the stability of SmSOD, the dimeric cambialistic superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans, towards temperature and guanidine hydrochloride. Thermal and chemical denaturations were investigated by means of circular dichroism, fourth-derivative UV spectroscopy and fluorescence measurements. Data indicate that SmSOD is endowed with a significant thermostability and that both its thermal and guanidine hydrochloride-induced unfolding processes occur through a three-state model, characterized by a catalytically active dimeric intermediate species. To our knowledge, SmSOD is the smallest known dimeric protein that populates a well-structured active dimeric rather than a monomeric intermediate during unfolding processes.     

Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles

The thermodynamic and spectroscopic properties of a cysteine-free variant of Escherichia coli dihydrofolate reductase (AS-DHFR) were investigated using the combined effects of urea and temperature as denaturing agents. Circular dichroism (CD), absorption, and fluorescence spectra were recorded during temperature-induced unfolding at different urea concentrations and during urea-induced unfolding at different temperatures. The first three vectors obtained by singular-value decomposition of each set of unfolding spectra were incorporated into a global analysis of a unique thermodynamic model. Although individual unfolding profiles can be described as a two-state process, a simultaneous fit of 99 vectors requires a three-state model as the minimal scheme to describe the unfolding reaction along both perturbation axes. The model, which involves native (N), intermediate (I), and unfolded (U) states, predicts a maximum apparent stability, DeltaG degrees (NU), of 6 kcal mol(-)(1) at 15 degrees C, an apparent m(NU) value of 2 kcal mol(-)(1) M(-)(1), and an apparent heat capacity change, DeltaC(p)()(-NU), of 2.5 kcal mol(-)(1) K(-)(1). The intermediate species has a maximum stability of approximately 2 kcal mol(-)(1) and a compactness closer to that of the native than to that of the unfolded state. The population of the intermediate is maximal ( approximately 70%) around 50 degrees C and falls below the limits of detection of > or =2 M urea or at temperatures of <35 or >65 degrees C. The fluorescence properties of the equilibrium intermediate resemble those of a transient intermediate detected during refolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster in the adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CD spectroscopic properties of the native state reveal the presence of two principal isoforms that differ in ligand binding affinities and in the packing of the adenosine-binding domain. The relative populations of these species change slightly with temperature and do not depend on the urea concentration, implying that the two native isoforms are well-structured and compact. Global analysis of data from multiple spectroscopic probes and several methods of unfolding is a powerful tool for revealing structural and thermodynamic properties of partially and fully folded forms of DHFR.     

Unfolding of class A amphipathic peptides on a lipid surface

The folding of polypeptides associated with biomembranes is a ubiquitous phenomenon, yet the thermodynamics underlying the process are poorly understood. In the present work we examine the unfolding of a series of alpha-helical amphipathic membrane-associated peptides using guanidine hydrochloride as a denaturant. The peptides are based on the class A amphipathic helix motif, and each contains a single tryptophan at sequence position 2, 3, 7, 12, or 14. The isothermal unfolding process was monitored by circular dichroism ellipticity at 222 nm to monitor changes in the helical structure of the peptide. Tryptophan fluorescence was used to probe the local changes in the environment about the indole fluorophore. The unfolding curves generated from the two experimental techniques for each peptide-lipid complex were non-coincidental, suggesting the presence of stable intermediate(s) in the unfolding. A three-state model could adequately account for the data and yielded parameters which were consistent with the presence of a partially folded intermediate structure which (i) is closer in Gibb's free energy to the folded state than the unfolded state and (ii) retains much of the interfacial and amphipathic character of the folded state. Denaturant-induced peptide dissociation from the peptide-lipid complexes was found to be negligible as confirmed by size exclusion chromatography. The results are compared with related thermodynamic data and discussed in terms of current models of peptide folding at membrane interfaces.     

Folding of Escherichia coli DsbC: characterization of a monomeric folding intermediate

The homodimeric protein DsbC is a disulfide isomerase and a chaperone located in the periplasm of Escherichia coli. We have studied the guanidine hydrochloride (GdnHCl)-induced unfolding and refolding of DsbC using mutagenesis, intrinsic fluorescence, circular dichroism spectra, size-exclusion chromatography, and sedimentation velocity analysis. The equilibrium refolding and unfolding of DsbC was thermodynamically reversible. The equilibrium folding profile measured by fluorescence excited at 280 nm exhibited a three-state transition profile with a stable folding intermediate formed at 0-2.0 M GdnHCl followed by a second transition at higher GdnHCl concentrations. Sedimentation velocity data revealed dissociation of the dimer to the monomer over the concentration range of the first transition (0-2.0 M). In contrast, fluorescence emission data for DsbC excited at 295 nm showed a single two-state transition. Fluorescence emission data for the equilibrium unfolding of the monomeric G49R mutant, excited at either 295 or 280 nm, indicated a single two-state transition. Data obtained for the dimeric Y52W mutant indicated a strong protein concentration dependence of the first transition but no dependence of the second transition in equilibrium unfolding. This suggests that the fluorescence of Y52W sensitively reports conformational changes caused by dissociation of the dimer. Thus, the folding of DsbC follows a three-state transition model with a monomeric folding intermediate formed in 0-2.0 M GdnHCl. The folding of DsbC in the presence of DTT indicates an important role for the non-active site disulfide bond in stabilizing the conformation of the molecule. Dimerization ensures the performance of chaperone and isomerase functions of DsbC.     

The effect of non-enzymatic glycation on the unfolding of human serum albumin

We monitored the unfolding of human serum albumin (HSA) and glycated human serum albumin (gHSA) subjected to guanidine hydrochloride (GndHCl) by using fluorescence and circular dichroism (CD) spectroscopy. A two-state model with sloping baselines best described the Trp-214 fluorescence unfolding measurements, while a three-state model best described the far-UV CD unfolding data. Glycation of HSA increased the [D](50%) point by approximately 0.20M. This corresponded to an increase in the free energy of unfolding of gHSA relative to HSA of 2.6kJ/mol. The intrinsic fluorescence of Trp-214 in gHSA is 0.72 of that of HSA and the far-UV CD spectrum of gHSA is nearly identical to that of HSA. These results showed that glycation altered the local structure around Trp-214 while not significantly impacting the secondary structure, and this alteration translated into an overall change in the stability of gHSA compared to HSA.     

The fluorescence detected guanidine hydrochloride equilibrium denaturation of wild-type staphylococcal nuclease does not fit a three-state unfolding model

A three-state equilibrium unfolding of a protein can be difficult to detect if two of the states fail to differ in some easily measurable way. It has been unclear whether staphylococcal nuclease unfolds in a two-state fashion, with only the native and denatured states significantly populated at equilibrium, or in a three-state manner, with a well-populated intermediate. Since equilibrium unfolding experiments are commonly used to determine protein stability and the course of denaturation are followed by changes in the fluorescence which has difficulty in distinguishing various states, this is a potential problem for many proteins. Over the course of twenty years we have performed more than one hundred guanidine hydrochloride equilibrium denaturations of wild-type staphylococcal nuclease; to our knowledge, a number of denaturations unrivaled in any other protein system. A careful examination of the data from these experiments shows no sign of the behavior predicted by a three-state unfolding model. Specifically, a three-state unfolding should introduce a slight, but characteristic, non-linearity to the plot of stability versus denaturant concentration. The average residuals from this large number of repeated experiments do not show the predicted behavior, casting considerable doubt on the likelihood of a three-state unfolding for the wild-type protein. The methods used for analysis here could be applied to other protein systems to distinguish a two-state from a three-state denaturation.     

Thermodynamic stability of human lens recombinant alphaA- and alphaB-crystallins

Lens alpha-crystallin is a 600-800-kDa heterogeneous oligomer protein consisting of two subunits, alphaA and alphaB. The homogeneous oligomers (alphaA- and alphaB-crystallins) have been prepared by recombinant DNA technology and shown to differ in the following biophysical/biochemical properties: hydrophobicity, chaperone-like activity, subunit exchange rate, and thermal stability. In this study, we studied their thermodynamic stability by unfolding in guanidine hydrochloride. The unfolding was probed by three spectroscopic parameters: absorbance at 235 nm, Trp fluorescence intensity at 320 nm, and far-UV circular dichroism at 223 nm. Global analysis indicated that a three-state model better describes the unfolding behavior than a two-state model, an indication that there are stable intermediates for both alphaA- and alphaB-crystallins. In terms of standard free energy (DeltaG(NU)(H(2)(O))), alphaA-crystallin is slightly more stable than alphaB-crystallin. The significance of the intermediates may be related to the functioning of alpha-crystallins as chaperone-like molecules.     

绿脓杆菌apoazurin在溶液中存在两种天然构象

如何解释绿脓杆菌apoazurin变性过程的复杂机制是一个有争议的问题.最近的研究表明apoazurin的复杂变性机制可以归结为其天然态存在着至少两种构象.利用内源荧光发射谱和圆二色谱进一步研究了apoazurin的脲变性机制,发现其稳态脲变性符合表观的二态过程,但其动力学为双相过程.在高浓度脲中快反应在几秒钟内完成,而慢反应要经过几个小时.快反应和慢反应的mu值分别为2.24和2.45kJ·mol-1·M-1,去折叠活化能的差值为22kJ·mol-1.时间分辨的荧光发射谱和圆二色谱可以用天然态和完全变性态的谱图通过一个固定的比例参数进行重建.结果表明,过去被广泛接受的存在着变性中间体的机制是不正确的,而apoazurin在天然态存在至少两种构象的假设是合理的.     

Kinetics of folding and unfolding of goat alpha-lactalbumin

The equilibrium unfolding and the kinetic folding and unfolding of goat alpha-lactalbumin (GLA) were studied by near- and far-ultraviolet circular dichroism (CD) and by stopped-flow fluorescence spectroscopy. Specifically, the influence of environmental conditions such as pH and Ca2+ binding was examined. Compared to the apo-form, the Ca2+-bound form was found to be strongly stabilized in equilibrium conditions at pH 7.5 and 25 degrees C. The kinetics of the refolding of apo-GLA show a major change of fluorescence intensity during the experimental dead-time, but this unresolved effect is strongly diminished in holo-GLA. In both cases, however, the chevron plots can adequately be fitted to a three-state model. Moreover, double-mix stopped-flow experiments showed that the native state (N) is reached through one major pathway without the occurrence of alternative tracks. In contrast to the homologous bovine alpha-lactalbumin (BLA), the compactness of GLA is strongly influenced by the presence of Ca2+ ions. Unlike the two-state transition observed in guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation experiments at higher pH, an equilibrium intermediate state (I) is involved in denaturation at pH 4.5. In the latter case, analysis of the kinetic data makes clear that the intermediate and the unfolded states (U) show practically no Gibbs free energy difference and that they are in rapid equilibrium with each other. A possible explanation for these variations in stability and in folding characteristics with pH could be the degree of protonation of His107 that directly influences non-native interactions. Variation of environmental conditions and even small differences in sequence, therefore, can result in important effects on thermodynamic and folding parameters.     

Reversible thermal denaturation of human FGF-1 induced by low concentrations of guanidine hydrochloride.

Human acidic fibroblast growth factor (FGF-1) is a powerful mitogen and angiogenic factor with an apparent melting temperature (Tm) in the physiological range. FGF-1 is an example of a protein that is regulated, in part, by stability-based mechanisms. For example, the low Tm of FGF-1 has been postulated to play an important role in the unusual endoplasmic reticulum-independent secretion of this growth factor. Despite the close relationship between function and stability, accurate thermodynamic parameters of unfolding for FGF-1 have been unavailable, presumably due to effects of irreversible thermal denaturation. Here we report the determination of thermodynamic parameters of unfolding (DeltaH, DeltaG, and DeltaCp) for FGF-1 using differential scanning calorimetry (DSC). The thermal denaturation is demonstrated to be two-state and reversible upon the addition of low concentrations of added guanidine hydrochloride (GuHCl). DeltaG values from the DSC studies are in excellent agreement with values from isothermal GuHCl denaturation monitored by fluorescence and circular dichroism (CD) spectroscopy. Furthermore, the results indicate that irreversible denaturation is closely associated with the formation of an unfolding intermediate. GuHCl appears to promote reversible two-state denaturation by initially preventing aggregation of this unfolding intermediate, and at subsequently higher concentrations, by preventing formation of the intermediate.     

Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.     

Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies.

Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.     

Cutinase unfolding and stabilization by trehalose and mannosylglycerate

The unfolding of cutinase at pH 4.5 was induced by increasing the temperature and guanidine hydrochloride concentration in the presence of potassium chloride, trehalose, and mannosylglycerate potassium salt. Protein thermal unfolding approached a two-state process, since the unfolding transitions were coincident within experimental error when assessed by near-ultraviolet (UV) difference, tryptophyl, and 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence spectroscopy. Trehalose at 0.5 M increased the temperature at which 50% of cutinase is unfolded by 3 degrees C. Unfolding induced by guanidine hydrochloride is clearly a non-two-state process. The presence of a stable intermediate was detected because unfolding assessed by near-UV difference spectroscopy occurs earlier than unfolding assessed by tryptophyl fluorescence. The intermediate is molten globule in character: the ANS fluorescence is higher than in the presence of the folded or unfolded state, showing native-like secondary structure and losing many tertiary interactions of the folded state, i.e., those surrounding the tyrosyl microenvironment. The stabilization effect of trehalose and mannosylglycerate was quantified by fitting the unfolding transitions to a model proposed by Staniforth et al. (Biochemistry 1993;32:3842-3851). This model takes into consideration the increase in solvation energies of the amino acid side-chains as the denaturant concentration was increased and the fraction of amino acid side-chains that become exposed in the unfolded structure of cutinase. Trehalose and mannosylglycerate stabilize the folded state relative to the intermediate by 1.4-1.6 and 1.6 kcal/mol and the intermediate relative to the unfolded state by 1.0 and 1.5 kcal/mol, respectively.     

Identification of an equilibrium intermediate in the unfolding process of galectin-1, which retains its carbohydrate-binding specificity

The unfolding process of galectin-1 (Gal-1) in the presence of a denaturing agent was examined using fluorescence and far-UV circular dichroism (CD) spectroscopy determinations, and was found to be completely reversible. The data showed that the transitions of guanidine hydrochloride (GdnHCl)-induced lectin unfolding, in the absence of ligand, were biphasic in nature, clearly showing the existence of at least one stable intermediate. On the other hand, the unfolding in the presence of disaccharide yielded data that could fit very well to a two-state model, indicating a stabilizing effect of the ligand. The folding intermediate was further characterized by size exclusion chromatography, near-UV CD and anilinonaphtalene sulfonate binding, and shown to belong to the molten globule type. Strikingly, this intermediate retained its carbohydrate-binding specificity, as evidenced by the tryptophan fluorescence changes detected upon its interaction with lactose.     

Chemical modification of tryptophan residues and stability changes in proteins

The role of tryptophan residues in the stability of proteins was studied by ozone oxidation, which causes a small change in the tryptophan side chain. Trp 187 of the constant fragment of a type lambda immunoglobulin light chain, Trp 59 of ribonuclease T1, and Trp 62 of hen egg white lysozyme were oxidized specifically by ozone to N'-formylkynurenine or kynurenine. Judging from their circular dichroic and fluorescence spectra, these modified proteins were found to be the same as those of the respective intact proteins. However, even the slight modification of a single tryptophan residue produced a large decrease in the stability of these proteins to guanidine hydrochloride and heat. The smaller the extent of exposure of the tryptophan residue, the greater the effect of the modification on the stability. The formal kinetic mechanism of unfolding and refolding by guanidine hydrochloride of the CL fragment was not altered by tryptophan oxidation, but the rate constants for unfolding and refolding changed. The thermal unfolding transitions were analyzed to obtain the thermodynamic parameters. The enthalpy and entropy changes for the modified proteins were larger than the respective values for the intact proteins.     

Folding of a de novo designed native-like four-helix bundle protein

The folding of a model native-like dimeric four-helix bundle protein, (alpha(2))(2), was investigated using guanidine hydrochloride, hydrostatic pressure, and low temperature. Unfolding by guanidine hydrochloride followed by circular dichroism and intrinsic fluorescence spectroscopy revealed a highly cooperative transition between the native-like and unfolded states, with free energy of unfolding determined from CD data, DeltaG(unf) = 14.3 +/- 0.8 kcal/mol. However, CD and intrinsic fluorescence data were not superimposable, indicating the presence of an intermediate state during the folding transition. To stabilize the folding intermediate, we used hydrostatic pressure and low temperature. In both cases, dissociation of the dimeric native-like (alpha(2))(2) into folded monomers (alpha(2)) was observed. van't Hoff analysis of the low temperature experiments, assuming a two-state dimer 171-monomer transition, yielded a free energy of dissociation of (alpha(2))(2) of DeltaG(diss) = 11.4 +/- 0.4 kcal/mol, in good agreement with the free energy determined from pressure dissociation experiments (DeltaG(diss) = 10.5 +/- 0.1 kcal/mol). Binding of the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to the pressure- and cold-dissociated states of (alpha(2))(2) indicated the existence of molten-globule monomers. In conclusion, we demonstrate that the folding pathway of (alpha(2))(2) can be described by a three-state transition including a monomeric molten globule-like state.     

Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.