Resveratrol induces thermal stabilization of human serum albumin and modulates the early aggregation stage.

Several phenolic compounds bind to proteins and show the ability to interfere with their aggregation process. The impact of the natural polyphenol resveratrol on the stability and heat induced aggregation of human serum albumin (HSA) was investigated by differential scanning calorimetry (DSC), attenuated total reflectance Fourier transform infrared (ATR-FTIR), UV-vis absorbance, ThT fluorescence, atomic force microscopy (AFM) and molecular modeling. The binding of resveratrol to HSA improves the stability of the protein to thermal unfolding, particularly for the energetic domain containing the ligand binding site, as modeled by computational techniques. The thermal unfolding is irreversible and after the melting the protein aggregates, either with or without the ligand. The kinetics of HSA aggregation between 70 and 80°C shows an exponential growth of the absorbance change and it slows down when resveratrol is added. The aggregates have fibril-like morphology and resveratrol attenuates the formation of ß-structured species. The overall results suggest that resveratrol stabilizes the protein structure and modulates the formation of fibrils along the initial stage of the HSA aggregation pathway.

Fatty acid binding into the highest affinity site of human serum albumin observed in molecular dynamics simulation.

Multiple molecular dynamics simulations were performed to investigate the association of stearic acid into the highest affinity binding site of human serum albumin. All binding events ended with a rapid (<10 ps) lock-in of the fatty acid due to formation of a hydrogen bond with Tyr401. The kinetics and energetics of the penetration process both depended linearly on the positional shift of the fatty acid, with an average insertion time and free energy reduction of, respectively, 32 ± 20 ps and 0.70 ± 0.15 kcal/mol per methylene group absorbed. Binding events of longer duration (tbind>1 ns) were characterized by a slow exploration of the pocket entry and, frequently, of a nearby protein crevice corresponding to a metastable state along the route to the binding site. Taken all together, these findings reconstruct the following pathway for the binding process of stearic acid: (i) contact with the protein surface, possibly facilitated by the presence of an intermediate location, (ii) probing of the site entry, (iii) insertion into the protein, and (iv) lock-in at the final position. This general description may also apply to other long-chain fatty acids binding into any of the high-affinity sites of albumin, or to specific sites of other lipid-binding proteins.

Chain interdigitation in DPPC bilayers induced by HgCl2: evidences from continuous wave and pulsed EPR.

Continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy and electron spin echo methods of pulsed EPR of phosphatidylcholine spin-labeled at different positions, n, in the sn-2 chain (n-PCSL, n=5, 7, 10, 12, 14, and 16) are used to study the interaction of inorganic mercury chloride HgCl2 with multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC). For temperatures through the gel phase of DPPC multilayers, the CW-EPR spectra show that an increase of HgCl2 content in the dispersion medium slightly increases the rotational mobility of 5-PCSL and markedly restricts the motion of 16-PCSL. Mercury chloride at 100mM (HgCl2/lipid molar ratio=2:1) removes the gradient of increasing mobility along the chain found in DPPC bilayers in the gel phase. In contrast, HgCl2 does not influence the DPPC chain flexibility profile in the fluid phase. It also suppresses the pre-transition and moderately downshifts the main transition temperature of DPPC membranes. These findings indicate that HgCl2 affects the lipid chain packing of DPPC bilayers and are consistent with the induction of an interdigitated gel phase. Further, D2O-electron spin echo envelope modulation spectroscopy indicates that in the interdigitated phase a higher water permeation is favored at any chain position and the sigmoidal transmembrane water accessibility profile of DPPC bilayers is abolished. Accordingly, the positional dependence of (14)N-hyperfine splitting, 2Azz, shows that the typical hydrophobic barrier of DPPC is significantly altered in the interdigitated phase and all the segments of the lipid chains result to be in a more polar environment.

Librational fluctuations in protein glasses.

Librational motions in the region of the protein "glass" (or dynamic) transition are analysed for spin-labelled haemoglobin, serum albumin and ß-lactoglobulin by EPR spectroscopy. A discontinuity in the temperature dependence of the mean-square librational amplitude, <α(2)>, occurs in the region of 200K as found for the mean-square atomic displacement, , at the protein dynamic transition by Mössbauer spectroscopy and neutron scattering. The discontinuity in <α(2)> vs. T can be described by the Vogel-Tammann-Fulcher equation, implying a finite glass transition temperature. Above the dynamic transition, <α(2)> vs. 1/T can be approximated by the Arrhenius law with activation energies similar to those usually found for , and relaxation processes in glass-forming media and the hydration shells of proteins. Similar results are found for librational fluctuations of membranous Na,K-ATPase spin-labelled either on superficial SH groups or on those essential to activity.

Influence of stearic acids on resveratrol-HSA interaction.

The interaction between the natural polyphenol resveratrol and human serum albumin (HSA), the most abundant transport protein in plasma, has been studied in the absence and in the presence of up to six molecules of stearic acids (SA) pre-complexed with the protein. The study has been carried out by using the intrinsic fluorescence of both HSA and resveratrol. Protein and polyphenol fluorescence data indicate that resveratrol binds to HSA with an association constant k(a) = (1.10 ± 0.14) × 10(5) M(-1) and (1.09 ± 0.02) × 10(5) M(-1), respectively, whereas Job plot evidences the formation of an equimolar protein/drug complex. Low SA content associated with HSA does not affect significantly the structural conformation of the protein and its interaction with resveratrol, whereas high SA content induces conformational changes in the protein, and reduces resveratrol binding affinity. The photostability of resveratrol in the different samples changes in the order: buffer < (high [SA]/HSA) < HSA < (low [SA]/HSA). The results on (SA/HSA)-resveratrol samples highlight the ability of the protein to bind hydrophobic and amphiphilic ligands and to protect from degradation an important antioxidant molecule under biologically relevant conditions.

The influence of active site loop mutations on the thermal stability of azurin from Pseudomonas aeruginosa.

The copper site and overall structures of azurin (AZ) variants in which the amicyanin (AMI) and plastocyanin (PC) metal binding loops have been introduced, AZAMI and AZPC, respectively, are similar to that of AZ, whereas the loop conformations resemble those in the native proteins. To assess the influence of these loop mutations on stability, the thermal unfolding of AZAMI and AZPC has been investigated by differential scanning calorimetry, absorption and fluorescence spectroscopy. The calorimetric profiles of both variants exhibit a complex shape consisting of two endothermic peaks and an exothermic peak. The temperature of the maximum heat of absorption for the single endothermic peak is 82.7°C for AZ, whereas for AZAMI and AZPC the most intense endothermic peaks are at 74.9 and 68.1°C comparable to values for AMI and PC, respectively. Denaturation investigated using the temperature dependence of the absorbance at ∼600nm and Trp emission, also demonstrates decreased stability for both loop mutants. The thermal transition between the native and the denaturated states is irreversible, scan rate dependent and consistent with the two-state irreversible model. The structure of the active-site loop has a dramatic effect on the kinetic stability and the unfolding pathway of cupredoxins.

Early stage aggregation of human serum albumin in the presence of metal ions.

The heat induced aggregation of human serum albumin (HSA) with and without an equimolar amount of Cu(II) and Zn(II) was investigated by using optical absorption, fluorescence, AFM and EPR spectroscopy. Turbidity experiments as a function of temperature indicate that the protein aggregation occurs after the melting of the protein. The kinetic of HSA aggregation, investigated between 60 and 70°C by monitoring the optical density changes at 400nm on a 180min time window, shows an exponential growth with a rate that increases with the temperature. Fluorescence of the thioflavin T evidences a significant increase of the intensity at 480nm at increasing incubation time. These results combined with AFM experiments show that the protein aggregates are elongated oligomers with fibrillar-like features. The absence of a lag-phase suggests that the early stage aggregation of HSA follows a downhill pathway that does not require the formation of an organized nucleus. The presence of Cu(II) and Zn(II) ions does not affect the thermally induced aggregation process and the morphology of HSA aggregates. The result is compatible with the binding of the metal ions to the protein in the native state and with the high conformational stability of HSA.

Spin-echo EPR of Na,K-ATPase unfolding by urea.

Denaturant-perturbation and pulsed EPR spectroscopy are combined to probe the folding of the membrane-bound Na,K-ATPase active transport system. The Na,K-ATPase enzymes from shark salt gland and pig kidney are covalently spin labelled on cysteine residues that either do not perturb or are essential to hydrolytic activity (Class I and Class II -SH groups, respectively). Urea increases the accessibility of water to the spin-labelled groups and increases their mutual separations, as recorded by D2O interactions from ESEEM spectroscopy and instantaneous spin diffusion from echo-detected EPR spectra, respectively. The greater effects of urea are experienced by Class I groups, which indicates preferential unfolding of the extramembrane domains. Conformational heterogeneity induced by urea causes dispersion in spin-echo phase-memory times to persist to higher temperatures. Analysis of lineshapes from partially relaxed echo-detected EPR spectra indicates that perturbation by urea enhances the amplitude and rate of fluctuations between conformational substates, in the higher temperature regime, and also depresses the glasslike transition in the protein. These non-native substates that are promoted by urea lie off the enzymatic pathway and contribute to the loss of function.

Solvent effect on librational dynamics of spin-labelled haemoglobin by ED- and CW-EPR.

Two-pulse, echo-detected electron paramagnetic resonance (ED-EPR) spectra and continuous-wave EPR (CW-EPR) spectra were used to investigate the solvent effect on the librational motion of human haemoglobin spin-labelled on cysteine ß93 with the nitroxide derivative of maleimide, 6-MSL. Protein samples fully hydrated in phosphate buffer solution (PBS), in a 60% v/v glycerol/water mixture and in the lyophilized form were measured at cryogenic temperature in the frozen state. The protein librational motion was characterized by the amplitude-correlation time product, <α²>τ(c), deduced from the ED-EPR spectra. The librational amplitude, <α²>τ(c), was determined independently, from the motionally averaged hyperfine splitting in the CW-EPR spectra, and the librational correlation time, τ(c), was derived from the combination of the pulsed and conventional EPR data. Rapid librational motion of small amplitude was detected in all samples. In each case, the librational dynamics was restricted up to 180 K, beyond which it increased steeply for the hydrated protein in PBS and in the presence of glycerol. In contrast, in the dehydrated protein, the librational dynamics was hindered and less dependent on temperature up to ~240 K. In all samples, <α²> deviated from small values only for T > 200 K, where a rapid increase of <α²> was evident for the hydrated samples, whereas limited temperature variation was shown in the lyophilized samples. The librational correlation time was in the sub-nanosecond regime and weakly dependent on temperature. The results evidence that solvent favours protein dynamics.

Spontaneous transfer of stearic acids between human serum albumin and PEG:2000-grafted DPPC membranes.

Electron spin resonance (ESR) spectroscopy is used to study the transfer of stearic acids between human serum albumin (HSA) and sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Protein/lipid dispersions are considered in which spin-labelled stearic acids at the 16th carbon atom along the acyl chain (16-SASL) are inserted either in the protein or in the SSL. Two component ESR spectra with different rotational mobility are obtained over a broad range of temperature and membrane composition. Indeed, superimposed to an anisotropic protein-signal, appears a more isotropic lipid-signal. Since in the samples only one matrix (protein or membranes) is spin-labelled, the other component accounts for the transfer of 16-SASL between albumin and membranes. The two components have been resolved and quantified by spectral subtractions, and the fraction, f (p) (16-SASL), of spin labels bound non-covalently to the protein has been used to monitor the transfer. It is found that it depends on the type of donor and acceptor matrix, on the physical state of the membranes and on the grafting density of the polymer-lipids. Indeed, it is favoured from SSL to HSA and the fraction of stearic acids transferred increases with temperature in both directions of transfer. Moreover, in the presence of polymer-lipids, the transfer from HSA to SSL is slightly attenuated, especially in the brush regime of the polymer-chains. Instead, the transfer from SSL to HSA is favoured by the polymer-lipids much more in the mushroom than in the brush regime.

Molecular dynamics of amicyanin reveals a conserved dynamical core for blue copper proteins.

Molecular dynamics simulation has been carried out for the blue copper protein amicyanin from two different sources, Paracoccus denitrificans and Paraccocus versutus, to investigate the structural and dynamical properties common to the two molecules and to identify prominent features shared with proteins of the same family, the monomeric cupredoxins. The two amicyanins have almost identical secondary and tertiary structure. In the simulation, they differ for the number of hydrogen bonds in the main chain and the conformation of some beta-strands. However, they strictly maintain the arrangement of the portions of the beta-barrel that are conserved in the folding architecture of the blue copper proteins. Paracoccus versutus amicyanin equilibrates more rapidly, shows lower atomic deviation values, and is less rigid with respect to Paracoccus denitrificans amicyanin. Principal component analysis reveals that the conformational subspaces corresponding to eigenvectors with the same index for each of the two molecules are not necessarily equivalent. Nevertheless, a core scaffold with constrained dynamics exist for both amicyanins. In addition, two fairly flexible regions that are located on the opposite side with respect to the interaction sites with the partner molecules in the redox process have been evidenced in the protein structure. This description of amicyanin, with a few mobile regions remote from the active site and a rigid scaffold including most of the protein beta-barrel, has a close similarity with that of azurin and plastocyanin, two other cupredoxins previously investigated in simulation. Furthermore, similarities in the distribution of the atomic fluctuations indicate that amicyanin, azurin, and plastocyanin possess common dynamical features, in spite of differences in their structure. On the basis of these findings, we suggest that topological constraints imposed by the folding in correspondence of protein regions that are the most conserved determine the protein dynamics of the cupredoxin family. The dynamical properties of the cupredoxins might be controlled for functional advantages that include the binding mechanism with the biological partners and the collective inner motions of the protein matrix required for the electron transfer, whereas long-range conformational changes in the redox reaction should be excluded.

Thermal unfolding studies of a phytocyanin.

The thermal stability of umecyanin, a stellacyanin from horseradish roots, has been investigated by differential scanning calorimetry, optical absorption and fluorescence spectroscopy at neutral and alkaline pH. Above pH 9 the Cu(II) protein experiences a blue shift of the main visible absorption band at approximately 600 nm and changes colour from blue to violet. The thermal transition of the protein is irreversible and occurs between 61.4 and 68.8 degrees C at pH 7.5 and between 50.7 and 57.4 degrees C at pH 9.8. The calorimetric data indicates that at both pH values the thermally induced transition of the protein between the native and denaturated states can be described in terms of the classical Lumry-Eyring unfolding model Native<-->Unfolded-->Final. The analysis of the reversible step in the unfolding pathway demonstrates a significant reduction in conformational stability (DeltaG) of the alkaline form of the protein. Such a reduction is consistent with an enhanced flexibility of UMC at high pH and has mainly entropic character.

Spectroscopic and calorimetric studies on the interaction of human serum albumin with DPPC/PEG:2000-DPPE membranes.

Site specific spectroscopic techniques and differential scanning calorimetry were used to study human serum albumin (HSA) in the absence and in the presence of membranes composed of dipalmitoylphosphatidylcholine (DPPC) and poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Electron spin resonance (ESR) of a maleimide spin-label (5-MSL) covalently bound to the free sulfhydryl group at the unique cystein Cys-34 in domain I, intrinsic fluorescence of the single tryptophan Trp-214 in domain II, and extrinsic fluorescence of p-nitrophenyl anthranilate conjugated with tyrosine Tyr-411 in domain III were employed to study HSA dispersions with or without polymer-grafted membranes. On adsorbing at the DPPC membrane surfaces, domain I assumes a more loosened conformation and partitioning of the spin-labelled protein between the aqueous phase and the interfacial region of lipid membranes is observed by ESR. Domain II and III undergo a local structural arrangement which leads Trp-214 and Tyr-411 to come closer and causes intrinsic fluorescence quenching. The influence of DPPC bilayers on HSA is characterized both by a decrease of the thermal unfolding enthalpy and by a slight increase of the transition temperature, Tt, of the protein. The lipid induced effects on HSA are progressively reduced on increasing the amounts of PEG:2000-DPPE mixed with DPPC from the mushroom regime to the brush regime. Primary protein adsorption at the lipid surfaces is abolished at 1 mol% of the polymer-lipid, whereas the secondary protein adsorption at the polymer-brush leads to a further increase of both transition enthalpy and Tt relative to the case of aqueous dispersions of HSA alone.

Backbone dynamics of alamethicin bound to lipid membranes: spin-echo electron paramagnetic resonance of TOAC-spin labels.

Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and that induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogs in which a selected Aib residue (at position n = 1, 8, or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in association with phosphatidylcholine bilayer membranes. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2tau. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.

The role played by the alpha-helix in the unfolding pathway and stability of azurin: switching between hierarchic and nonhierarchic folding.

The role played by the alpha-helix in determining the structure, the stability and the unfolding mechanism of azurin was addressed by studying a helix-depleted azurin variant produced by site-directed mutagenesis. The protein structure was investigated by CD, 1D (1)H NMR, fluorescence spectroscopy measurements and MD simulations, whilst EPR, UV-visible and cyclic voltammetry experiments were carried out to investigate the geometry and the properties of the Cu(II) site. The effects of the alpha-helix depletion on the thermal stability and the unfolding pathway of the protein were determined by DSC, UV/visible and fluorescence measurements at increasing temperature. The results show that, in the absence of the alpha-helix segment, the overall protein structure is maintained, and that only the Cu site is slightly modified. In contrast, the protein stability is diminished by about 60% with respect to the wild-type azurin. Moreover, the unfolding pathway of the mutant azurin involves the presence of detectable intermediates. In comparison with previous studies concerning other small beta-sheet cupredoxins, the results as a whole support the hypothesis that the presence of the alpha-helix can switch the folding of azurin from a hierarchic to a nonhierarchic mechanism in which the highly conserved beta-sheet core provides a scaffold for cooperative folding of the wild-type protein.

Thermal stability effects of removing the type-2 copper ligand His306 at the interface of nitrite reductase subunits.

Nitrite reductase (NiR) is a highly stable trimeric protein, which denatures via an intermediate, N(3)<--(k)-->U(3)--(k)-->F (N-native, U-unfolded and F-final). To understand the role of interfacial residues on protein stability, a type-2 copper site ligand, His306, has been mutated to an alanine. The characterization of the native state of the mutated protein highlights that this mutation prevents copper ions from binding to the type-2 site and eliminates catalytic activity. No significant alteration of the geometry of the type-1 site is observed. Study of the thermal denaturation of this His306Ala NiR variant by differential scanning calorimetry shows an endothermic irreversible profile, with maximum heat absorption at T (max) approximately equal to 85 degrees C, i.e., 15 degrees C lower than the corresponding value found for wild-type protein. The reduction of the protein thermal stability induced by the His306Ala replacement was also shown by optical spectroscopy. The denaturation pathway of the variant is compatible with the kinetic model N(3)--(k)-->F(3), where the protein irreversibly passes from the native to the final state. No evidence of subunits' dissociation has been found within the unfolding process. The results show that the type-2 copper sites, situated at the interface of two monomers, significantly contribute to both the stability and the denaturation mechanism of NiR.

Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin.

Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D(2)O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature. Water penetration into the membrane is seen in the D(2)O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.

Structural, dynamical and functional aspects of the inner motions in the blue copper protein azurin.

Molecular dynamics was applied to dissect out the internal motions of azurin, a copper protein performing electron transfer. Simulations of 16.5 ns were analyzed in search of coordinated displacements of amino acid residues that are important for the protein function. A region with high conformational instability was found in the 'southern' end of the molecule, far away from the copper site and the binding sites for the redox partners of azurin. By excluding the 'southern' region from the subsequent analysis, correlated motions were identified in the hydrophobic patch that surrounds the protein active site. The simulation results are in excellent agreement with recent NMR data on azurin in solution [A. V. Zhuravleva, D. M. Korzhnev, E. Kupce, A. S. Arseniev, M. Billeter, V. Y. Orekhov, Gated electron transfers and electron pathways in azurin: a NMR dynamic study at multiple fields and temperatures, J. Mol. Biol. 342 (2004) 1599-1611] and suggest a rationale for cooperative displacements of protein residues that are thought to be critical for the electron transfer process. A number of other structural and dynamic features of azurin are discussed in the context of the blue copper protein family and an explanation is proposed to account for the variability/conservation of some regions in the cupredoxins.

Pro-oxidant activity of oleuropein determined in vitro by electron spin resonance spin-trapping methodology.

In this work, the pro-oxidant behavior of oleuropein (OLP, 1) is characterized in a Fenton-like experiment by means of ESR spectroscopy using the spin trap system DMSO and 4-(pyridyl-1-oxide)-N-tert-butyl nitrone (POBN) in phosphate buffer (PB) solution. Ferrous ions in the absence of hydrogen peroxide cause the formation of the stable nitroxide species 4 and 5 through the intermediate perferryl species. OLP displays its antioxidant activity in vitro blocking the oxidation path that leads to methoxyl radicals hence to the formation of the stable radical species 5. The role of the catechol moiety was proved when the perferryl experiments were repeated in the presence of the dimethylated oleuropein homologue (OLP-Met2, 2). The dual behavior of oleuropein, similar to that ascertained for other catechol and non-catechol natural active species, should provide warnings for its use as nutraceutical or as drug with manifold healing effects.

A comparative investigation of the thermal unfolding of pseudoazurin in the Cu(II)-holo and apo form.

The contribution of the copper ion to the stability and to the unfolding pathway of pseudoazurin was investigated by a comparative analysis of the thermal unfolding of the Cu(II)-holo and apo form of the protein. The unfolding has been followed by calorimetry, fluorescence, optical density, and electron paramagnetic resonance (EPR) spectroscopy. The thermal transition of Cu(II)-holo pseudoazurin is irreversible and occurs between 60.0 and 67.3 degrees C, depending on the scan rate and technique used. The denaturation pathway of Cu(II)-holo pseudoazurin can be described by the Lumry-Eyring model: N --> U --> [corrected] F; the protein reversibly goes from the native (N) to the unfolded (U) state, and then irreversibly to the final (F) state. The simulation of the experimental calorimetric profiles, according to this model, allowed us to determine the thermodynamic and kinetic parameters of the two steps. The DeltaG value calculated for the Cu(II)-holo pseudoazurin is 39.2 kJ.mol(-1) at 25 degrees C. The sequence of events in the denaturation process of Cu(II)-holo pseudoazurin emergence starts with the disruption of the copper site and the hydrophobic core destabilization followed by the global protein unfolding. According to the EPR findings, the native type-1 copper ion shows type-2 copper features after the denaturation. The removal of the copper ion (apo form) significantly reduces the stability of the protein as evidenced by a DeltaG value of 16.5 kJ.mol(-1) at 25 degrees C. Moreover, the apo Paz unfolding occurs at 41.8 degrees C and is compatible with a two-state reversible process N --> [corrected] U.