pH-dependent urea-induced unfolding of stem bromelain: Unusual stability against urea at neutral pH

Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a function of pH using circular dichroism and fluorescence emission spectroscopy. Urea-induced denaturation studies at pH 4.5 showed that SB unfolds through a two-state mechanism and yields ΔG (free energy difference between the fully folded and unfolded forms) of ∼5.0 kcal/mol and C _m (midpoint of the unfolding transition) of ∼6.5 M at 25°C. Very high concentration of urea (9.5 M) provides unusual stability to the protein with no more structural loss and transition to a completely unfolded state.     

A nondetergent sulfobetaine improves protein unfolding reversibility in microcalorimetric studies

A nondetergent sulfobetaine (NDSB) was found to improve unfolding reversibility of several proteins by inhibiting heat-induced aggregation. As a consequence, ΔH_cal/ΔH_vH ratios were also improved to values close to 1 for a two-state unfolding. NDSB is effective in a wide range of pH values and especially at acidic pH generally used to calculate ΔC_p values by the Kirchhoff relation. The sulfobetaine also allows recording protein refolding by protecting the heat-induced unfolded state against aggregation.     

Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi

In this work, we explored the acid-induced unfolding pathway of non-porin outer membrane protein (OMP), an immunogenic protein from Salmonella Typhi, by monitoring the conformational changes over a pH range of 1.0–7.0 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, and dynamic light scattering. The spectroscopic measurements showed that OMP in its native state at pH 7.0 exists in more stable and compact conformation. In contrast, at pH 2.0, OMP retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii, and nearly four-fold increase in ANS fluorescence with respect to the native state, indicating that MG state exists at pH 2.0. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of a partially unfolded state between native and unfolded state. The effect of pH on the conformation and thermostability of OMP points towards its heat resistance at neutral pH (T _m?~?69 °C at pH 7.0, monitored by change in MRE_{222 nm}). Acid unfolded state was also characterized by the lack of a cooperative thermal transition. All these results suggested that acid-induced unfolded state of OMP at pH 2.0 represented the molten globule state. The chemical denaturation studies with GuHCl and urea as denaturants showed dissimilar results. The chemical unfolding experiments showed that in both far-UV CD and fluorescence measurements, GuHCl is more efficient than urea. GuHCl is characterized by low C _m (~1 M), while urea is characterized by high C _m (~3 M). The fully unfolded states were reached at 2 M GuHCl and 4 M urea concentration, respectively. This study adds to several key considerations of importance in the development of therapeutic agents against typhoid fever for clinical purposes.     

Helix-coil transition of Poly(L-glutamic acid) in N-methylacetamide

The folding of randomly coiled poly(L -glutamic acid) to the helical state has been studied in N-methylacetamide by titration methods. Since this solvent would be expected to form amide-peptide group hydrogen bonds with the unfolded form of the polymer, to a first approximation no helix stabilization could come from intrapolymer hydrogen bonds. The titration data, collected from 30 to 70°C yield the following values per residue for the thermodynamic parameters governing the coil-helix reaction for the uncharged polymer: ΔG_30°C°, ?1. 9 ± 0.1 kcal; Δ H°, 0 ± 0.1 kcal; ΔS_30°C°, 6.3 ± 0.6 eu. In N-methyl acetamide, the helix is an order of magnitude more stable than in water, and this stabilization appears to be entirely the result of the entropy gained by solvent molecules which are released from the polymer upon folding.     

Equilibrium unfolding studies of monellin: the double-chain variant appears to be more stable than the single-chain variant

To improve our understanding of the contributions of different stabilizing interactions to protein stability, including that of residual structure in the unfolded state, the small sweet protein monellin has been studied in both its two variant forms, the naturally occurring double-chain variant (dcMN) and the artificially created single-chain variant (scMN). Equilibrium guanidine hydrochloride-induced unfolding studies at pH 7 show that the standard free energy of unfolding, ΔG°(U), of dcMN to unfolded chains A and B and its dependence on guanidine hydrochloride (GdnHCl) concentration are both independent of protein concentration, while the midpoint of unfolding has an exponential dependence on protein concentration. Hence, the unfolding of dcMN like that of scMN can be described as two-state unfolding. The free energy of dissociation, ΔG°(d), of the two free chains, A and B, from dcMN, as measured by equilibrium binding studies, is significantly lower than ΔG°(U), apparently because of the presence of residual structure in free chain B. The value of ΔG°(U), at the standard concentration of 1 M, is found to be ～5.5 kcal mol(-1) higher for dcMN than for scMN in the range from pH 4 to 9, over which unfolding appears to be two-state. Hence, dcMN appears to be more stable than scMN. It seems that unfolded scMN is stabilized by residual structure that is absent in unfolded dcMN and/or that native scMN is destabilized by strain that is relieved in native dcMN. The value of ΔG°(U) for both protein variants decreases with an increase in pH from 4 to 9, apparently because of the thermodynamic coupling of unfolding to the protonation of a buried carboxylate side chain whose pK(a) shifts from 4.5 in the unfolded state to 9 in the native state. Finally, it is shown that although the thermodynamic stabilities of dcMN and scMN are very different, their kinetic stabilities with respect to unfolding in GdnHCl are very similar.     

Thermodynamic parameters of stabilization of the Yersinia pestis Caf113-149 subunit in compact form

Several experimental methods (circular dichroism, viscosity, intrinsic fluorescence, and fluorescence labeling) were used to study the conformational folding/unfolding transitions in a compact monomeric form of the Caf1_13-149 subunit under the action of guanidine hydrochloride in the temperature range 5–45°C. It has been shown that transitions always occur between two major states (unfolded and compact). This has made it possible to determine all the main thermodynamic functions that characterize the compact state of the Caf1_13-149 subunit: stability temperature T _m, free energy of stabilization ΔG _st, enthalpy ΔH _tr, and heat capacity jump ΔC in collapse of the structure. These data have been confirmed by an independent experiment on melting of fluorescently labeled protein.     

Effect of circular permutations on transient partial unfolding in proteins

Under native conditions, proteins can undergo transient partial unfolding, which may cause proteins to misfold or aggregate. A change in sequence connectivity by circular permutation may affect the energetics of transient partial unfolding in proteins without altering the three‐dimensional structures. Using Escherichia coli dihydrofolate reductase (DHFR) as a model system, we investigated how circular permutation affects transient partial unfolding in proteins. We constructed three circular permutants, CP18, CP37, and CP87, with the new N‐termini at residue 18, 37, and 87, respectively, and probed transient partial unfolding by native‐state proteolysis. The new termini in CP18, CP37, and CP87 are within, near, and distal to the Met20 loop, which is known to be dynamic and also part of the region that undergoes transient unfolding in wild‐type DHFR. The stabilities of both native and partially unfolded forms of CP18 are similar to those of wild‐type DHFR, suggesting that the influence of introducing new termini in a dynamic region to the protein is minimal. CP37 has a significantly more accessible partially unfolded form than wild‐type DHFR, demonstrating that introducing new termini near a dynamic region may promote transient partial unfolding. CP87 has significantly destabilized native and partially unfolded forms, confirming that modification of the folded region in a partially unfolded form destabilizes the partially unfolded form similar to the native form. Our findings provide valuable guidelines to control transient partial unfolding in designing circular permutants in proteins.     

Sequence‐dependent folding and unfolding of ligand‐bound purine riboswitches

Riboswitch regulation of gene expression requires ligand‐mediated RNA folding. From the fluorescence lifetime distribution of bound 2‐aminopurine ligand, we resolve three RNA conformers (C_o, C_i, C_c) of the liganded G‐ and A‐sensing riboswitches from Bacillus subtilis. The ligand binding affinities, and sensitivity to Mg²⁺, together with results from mutagenesis, suggest that C_o and C_i are partially unfolded species compromised in key loop‐loop interactions present in the fully folded C_c. These data verify that the ligand‐bound riboswitches may dynamically fold and unfold in solution, and reveal differences in the distribution of folded states between two structurally homologous purine riboswitches: Ligand‐mediated folding of the G‐sensing riboswitch is more effective, less dependent on Mg²⁺, and less debilitated by mutation, than the A‐sensing riboswitch, which remains more unfolded in its liganded state. We propose that these sequence‐dependent RNA dynamics, which adjust the balance of ligand‐mediated folding and unfolding, enable different degrees of kinetic discrimination in ligand binding, and fine‐tuning of gene regulatory mechanisms. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 953–965, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Equilibrium unfolding of class pi glutathione S-transferase

The equilibrium unfolding transition of class pi glutathione S-transferase, a homodimeric protein, from porcine lung was monitored by spectroscopic methods (fluorescence emission and ultraviolet absorption), and by enzyme activity changes. Solvent (guanidine hydrochloride and urea)-induced denaturation is well described by a two-state model involving significant populations of only the folded dimer and unfolded monomer. Neither a folded, active monomeric form nor stable unfolding intermediates were detected. The conformational stability, delta Gu (H2O), of the native dimer was estimated to be about 25.3 +/- 2 kcal/mol at 20 degrees C and pH6.5.     

Microsecond simulations of the folding/unfolding thermodynamics of the Trp‐cage miniprotein

We study the unbiased folding/unfolding thermodynamics of the Trp‐cage miniprotein using detailed molecular dynamics simulations of an all‐atom model of the protein in explicit solvent using the Amberff99SB force field. Replica‐exchange molecular dynamics simulations are used to sample the protein ensembles over a broad range of temperatures covering the folded and unfolded states at two densities. The obtained ensembles are shown to reach equilibrium in the 1 μs/replica timescale. The total simulation time used in the calculations exceeds 100 μs. Ensemble averages of the fraction folded, pressure, and energy differences between the folded and unfolded states as a function of temperature are used to model the free energy of the folding transition, ΔG(P, T), over the whole region of temperatures and pressures sampled in the simulations. The ΔG(P, T) diagram describes an ellipse over the range of temperatures and pressures sampled, predicting that the system can undergo pressure‐induced unfolding and cold denaturation at low temperatures and high pressures, and unfolding at low pressures and high temperatures. The calculated free energy function exhibits remarkably good agreement with the experimental folding transition temperature (T_f = 321 K), free energy, and specific heat changes. However, changes in enthalpy and entropy are significantly different than the experimental values. We speculate that these differences may be due to the simplicity of the semiempirical force field used in the simulations and that more elaborate force fields may be required to describe appropriately the thermodynamics of proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The interaction of collagen with the hydrophobic fluorescent probe-2-p-toluidinylnaphthalene-6-sulfonate.

Three kinds of fluorescence enhancement result from the interaction of 2-p-toluidinylnaphthalene-6-sulfonate and calf-skin collagen. They are negatively cooperative, independent, and highly cooperative fluorescence enhancement. In the independent region at pH 3.7, the binding number is about 36 moles of 2-p-toluidinylnaphthalene-6-sulfonate per mole of tropocollagen with a binding constant of 2.0 × 10⁴ M^?1; with ΔG = ?5.7 kcal/mole, ΔH = ?4.0 kcal/mole, and ΔS = 6 e.u. The pH dependence of fluorescence of native collagen shows that the deprotonated forms of the β and γ carboxyl groups of aspartic and glutamic acid decrease the intensity, possibly by charge repulsion of the negatively charged sulfonate group of 2-p-toluidinylnaphthalene-6-sulfonate. The positive charge of lysine is found to be unimportant in the interaction of 2-p-toluidinylnaphthalene-6-sulfonate with collagen. Fluorescence enhancement is caused mainly by the hydrophobic interactions of 2-p-toluidinylnaphthalene-6-sulfonate and collagen. Salt bridge formation between basic and acidic side chains in very low salt concentration may be detectable by 2-p-toluidinylnaphthalene-6-sulfonate fluorescence.     

Salt bridge as a gatekeeper against partial unfolding

Salt bridges are frequently observed in protein structures. Because the energetic contribution of salt bridges is strongly dependent on the environmental context, salt bridges are believed to contribute to the structural specificity rather than the stability. To test the role of salt bridges in enhancing structural specificity, we investigated the contribution of a salt bridge to the energetics of native‐state partial unfolding in a cysteine‐free version of Escherichia coli ribonuclease H (RNase H*). Thermolysin cleaves a protruding loop of RNase H^* through transient partial unfolding under native conditions. Lys86 and Asp108 in RNase H^* form a partially buried salt bridge that tethers the protruding loop. Investigation of the global stability of K86Q/D108N RNase H^* showed that the salt bridge does not significantly contribute to the global stability. However, K86Q/D108N RNase H^* is greatly more susceptible to proteolysis by thermolysin than wild‐type RNase H^* is. The free energy for partial unfolding determined by native‐state proteolysis indicates that the salt bridge significantly increases the energy for partial unfolding by destabilizing the partially unfolded form. Double mutant cycles with single and double mutations of the salt bridge suggest that the partially unfolded form is destabilized due to a significant decrease in the interaction energy between Lys86 and Asp108 upon partial unfolding. This study demonstrates that, even in the case that a salt bridge does not contribute to the global stability, the salt bridge may function as a gatekeeper against partial unfolding that disturbs the optimal geometry of the salt bridge.     

Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.     

Denaturation thermodynamics of chicken cardiac metmyoglobin

The unfolding at pH 8 of chicken cardiac aquometmyoglobin was examined as a function of temperature and concentration of guanidinium chloride using the two-state model. The isothermal unfolding data at 25°C were fitted to Tanford's transfer model and the binding model of Aune and Tanford. The estimates obtained for ΔG_D) were virtually identical, viz., 8.3 ±0.3 kcal mol^?1. The chicken metmyoglobin is thus some 5.3 kcal mol^?1 less stable than that of sperm whale metmyoglobin. The unfolding parameters α and Δn were decreased 20% from those of mammalian myoglobins thus far examined, suggesting nonidentity of native conformations. The apparent enthalpy change on unfolding was dependent on both temperature and denaturant concentration. The decreases in the isothermal unfolding parameters from those of sperm whale are principally assigned to three of the 46 sequence changes.     

Temperature‐induced partially unfolded state of hUBF HMG Box‐5: Conformational and dynamic investigations of the Box‐5 thermal intermediate ensemble

Human upstream binding factor (hUBF) HMG Box‐5 is a highly conserved protein domain, containing 84 amino acids and belonging to the family of the nonspecific DNA‐binding HMG boxes. Its native structure adopts a twisted L shape, which consists of three α‐helices and two hydrophobic cores: the major wing and the minor wing. In this article, we report a reversible three‐state thermal unfolding equilibrium of hUBF HMG Box‐5, which is investigated by differential scanning calorimetry (DSC), circular dichroism spectroscopy, fluorescence spectroscopy, and NMR spectroscopy. DSC data show that Box‐5 unfolds reversibly in two separate stages. Spectroscopic analyses suggest that different structural elements exhibit noncooperative transitions during the unfolding process and that the major form of the Box‐5 thermal intermediate ensemble at 55°C shows partially unfolded characteristics. Compared with previous thermal stability studies of other boxes, it appears that Box‐5 possesses a more stable major wing and two well separated subdomains. NMR chemical shift index and sequential ¹H^N_i‐¹H^N_i+1 NOE analyses indicate that helices 1 and 2 are native‐like in the thermal intermediate ensemble, while helix 3 is partially unfolded. Detailed NMR relaxation dynamics are compared between the native state and the intermediate ensemble. Our results implicate a fluid helix‐turn‐helix folding model of Box‐5, where helices 1 and 2 potentially form the helix 1‐turn‐helix 2 motif in the intermediate, while helix 3 is consolidated only as two hydrophobic cores form to stabilize the native structure. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Analysis of the ionization constants and heats of ionization of reduced and oxidized horse heart cytochrome c

Simultaneous curve fitting for the ionization parameters of oxidized and reduced horse heart cytochrome c in 0.15M KCl and 20°C yields values for the ionization constants (as pK′) and the heats of ionization (ΔH_i) which can reconstruct either the potentiometric or thermal titration curves. Reduced cytochrome c requires 8 sets of groups, whereas oxidized cytochrome c requires 10 sets of groups. The additional groups in the oxidized preparation appear to involve the ferriheme (pK′, 9.25; ΔH_i, 13.7 kcal/mol) and a tyrosine (pK′ ? 10.24) that is not present in the reduced form. The potentiometric and thermal difference curves (reduced – oxidized) involve the appearance of 17 kcal/mol centered at pH 9.7 and 5.8 kcal/mol centered at pH 4.9. The carboxyl groups in both species appear to be normal for the hydrogen-bonded form. Only one histidine has normal ionization properties (pK′, 6.7; ΔH_i, 7.5 kcal/mol), as do 17 of the lysine residues (pK′, 10.8; ΔH_i, 11.5 kcal/mol).     

Rates of structural fluctuations of lysozyme in the range of thermal unfolding transition

The hydrogen–deuterium exchange reaction for the tryptophan residues in lysozyme have been followed in 4.5M LiBr at pH 7.2 in the temperature range of the unfolding transition by measuring the transmittance change at 293 nm. The exchange reaction proceeded in three phases at low temperature for native protein. The first and the second phases were ascribed to the H-D exchange reactions of three relatively exposed tryptophan residues on the molecular surface. The third phase corresponded to the H-D exchange reaction of the three tryptophan residues buried in the interior of the molecule. The H-D exchange reaction proceeded in two phases near the melting temperature and in a single phase at high temperature, where almost all molecules are unfolded. The H-D exchange of three tryptophan residues buried in folded molecules was caused by fluctuation between the folded and unfolded structure of the protein molecule. The rates of such a fluctuation were determined from the rates of the exchange reaction at various temperatures. These rates agreed very well with those determined from the temperature-jump method. This means that a protein molecule in solution fluctuates between the N- and D-states at every temperature within the transition region, where the N-form is the tightly folded native structure and the D-form the randomly coiled chain. From measurements of thermal unfolding of ester-108-lysozyme and the binding constant of (NAG)₃ to ester-108-lysozyme, it was found that almost all cross-linked molecules are in the folded state near 50°C and pH 7.2 in 4.5M LiBr, where intact molecules are unfolded. We also studied the H-D exchange reaction of ester-108-lysozyme. In the temperature region of 43–50°C, about 70% of the exchangeable tryptophan residues of ester-108-lysozyme were exchanged within 1 s immediately after the mixing of D₂O, in spite of the fact that almost all molecules are in the folded state. This was considered the premelting of the surface of a corss-linked molecule.     

Unfolding of CBP21, a lytic polysaccharide monooxygenase,without dissociation of its copper ion cofactor

Chitin-binding protein 21 (CBP21) from Serratia marcescens is a lytic polysaccharide monooxygenase that contains a copper ion as a cofactor. We aimed to elucidate the unfolding mechanism of CBP21 and the effects of Cu²⁺ on its structural stability at pH 5.0. Thermal unfolding of both apo- and holoCBP21 was reversible. ApoCBP21 unfolded in a simple two-state transition manner. The peak temperature of the DSC curve, t_p, for holoCBP21 (74.4°C) was about nine degrees higher than that for apoCBP21 (65.6°C). The value of t_p in the presence of excess Cu²⁺ was around 75°C, indicating that Cu²⁺ does not dissociate from the protein molecule during unfolding. The unfolding mechanism of holoCBP21 was considered to be as follows: N∙Cu²⁺ ⇌ U∙Cu²⁺, where N and U represent the native and unfolded states, respectively. Urea-induced equilibrium unfolding analysis showed that holoCBP21 was stabilized by 35 kJ mol⁻¹ in terms of the Gibbs energy change for unfolding (pH 5.0, 25°C), compared with apoCBP21. The increased stability of holoCBP21 was considered to result from the structural stabilization of the protein-Cu²⁺ complex itself.     

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.