Urea-unfolded State Research Articles

Structural plasticity of T4 transcription co-activator gp33 revealed by a protease-resistant unfolded state

Gene 33 protein (gp33) is a transcriptional coactivator for late genes of the T4 bacteriophage. gp33 possesses a 5-helix bundle core, with unstructured N- and C-terminal regions that account for >50% of the protein sequence. It plays a unique role of interacting with host RNA polymerase, couples transcription with DNA replication, and plays the dual function as repressor and co-activator in phage transcription. Here, we identify protein structural plasticity as the molecular basis of the dual nature in gp33. We find that gp33 has the peculiar property of remaining protease insensitive in its urea-unfolded state. Using NMR studies with spectroscopic measurements, we propose that intra-protein interactions are replaced by protein-urea interactions in gp33. This process not only unfolds gp33 but also renders it protease-resistant. Our studies shed new light on the unique structural malleability of gp33 that might be important in its transition from a repressor to a late transcription co-activator.

The Folding process of Human Profilin-1, a novel protein associated with familial amyotrophic lateral sclerosis.

Human profilin-1 is a novel protein associated with a recently discovered form of familial amyotrophic lateral sclerosis. This urges the characterization of possible conformational states, different from the fully folded state, potentially able to initiate self-assembly. Under native conditions, profilin-1 is monomeric and possesses a well-defined secondary and tertiary structure. When incubated at low pH or with high urea concentrations, profilin-1 remains monomeric but populates unfolded states exhibiting larger hydrodynamic radius and disordered structure, as assessed by dynamic light scattering, far-UV circular dichroism and intrinsic fluorescence. Refolding from the urea-unfolded state was studied at equilibrium and in real-time using a stopped-flow apparatus. The results obtained with intrinsic fluorescence and circular dichroism indicate a single phase without significant changes of the corresponding signals before the major refolding transition. However, such a transition is preceded by a burst phase with an observed increase of ANS fluorescence, which indicates the conversion into a transiently populated collapsed state possessing solvent-exposed hydrophobic clusters. Kinetic analysis reveals that such state has a conformational stability comparable to that of the fully unfolded state. To our knowledge, profilin-1 is the first example of an amyloid-related protein where folding occurs in the absence of thermodynamically stable partially folded states.

Protein Adsorption into Mesopores: A Combination of Electrostatic Interaction, Counterion Release, and van der Waals Forces

Bovine heart cytochrome c has been immobilized into the mesoporous silica host material SBA-15 in both its native folded and urea-unfolded state. The comparison of the two folding states' behavior casts doubt on the commonly used explanation of cytochrome c adsorption, that is, the electrostatic interaction model. A detailed investigation of the protein binding as a function of pH and ionic strength of the buffer solution reveals the complex nature of the protein-silica interaction. Electrostatic interaction, van der Waals forces, and entropic contributions by counterion release each contribute to adsorption on the silica pore walls.

Denatured State Ensembles with the Same Radii of Gyration Can Form Significantly Different Long-Range Contacts

Defining the structural, dynamic, and energetic properties of the unfolded state of proteins is critical for an in-depth understanding of protein folding, protein thermodynamics, and protein aggregation. Here we analyze long-range contacts and compactness in two apparently fully unfolded ensembles of the same protein: the acid unfolded state of the C-terminal domain of ribosomal protein L9 in the absence of high concentrations of urea as well as the urea unfolded state at low pH. Small angle X-ray scattering reveals that the two states are expanded with values of Rg differing by <7%. Paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance studies, however, reveal that the acid unfolded state samples conformations that facilitate contacts between residues that are distant in sequence while the urea unfolded state ensemble does not. The experimental PRE profiles for the acid unfolded state differ significantly from these predicted using an excluded volume limit ensemble, but these long-range contacts are largely eliminated by the addition of 8 M urea. The work shows that expanded unfolded states can sample very different distributions of long-range contacts yet still have similar radii of gyration. The implications for protein folding and for the characterization of unfolded states are discussed.

Microsecond Subdomain Folding in Dihydrofolate Reductase

The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼ 550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR—in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse—emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed.

Evidence for Initial Non-specific Polypeptide Chain Collapse During the Refolding of the SH3 Domain of PI3 Kinase

Refolding of the SH3 domain of PI3 kinase from the guanidine hydrochloride (GdnHCl)-unfolded state has been probed with millisecond (stopped flow) and sub-millisecond (continuous flow) measurements of the change in fluorescence, circular dichroism, ANS fluorescence and three-site fluorescence resonance energy transfer (FRET) efficiency. Fluorescence measurements are unable to detect structural changes preceding the rate-limiting step of folding, whereas measurements of changes in ANS fluorescence and FRET efficiency indicate that polypeptide chain collapse precedes the major structural transition. The initial chain collapse reaction is complete within 150 μs. The collapsed form at this time possesses hydrophobic clusters to which ANS binds. Each of the three measured intra-molecular distances has contracted to an extent predicted by the dependence of the FRET signal in completely unfolded protein on denaturant concentration, indicating that contraction is non-specific. The extent of contraction of each intra-molecular distance in the collapsed product of sub-millisecond folding increases continuously with a decrease in [GdnHCl]. The gradual contraction is continuous with the gradual contraction seen in completely unfolded protein, and its dependence on [GdnHCl] is not indicative of an all-or-none collapse reaction. The dependence of the extent of contraction on [GdnHCl] was similar for the three distances, indicating that chain collapse occurs in a synchronous manner across different segments of the polypeptide chain. The sub-millisecond measurements of folding in GdnHCl were unable to determine whether hydrophobic cluster formation, probed by ANS fluorescence measurement, precedes chain contraction probed by FRET. To determine whether hydrogen bonding plays a role in initial chain collapse, folding was initiated by dilution of the urea-unfolded state. The extent of contraction of at least one intra-molecular distance in the collapsed product of sub-millisecond folding in urea is similar to that seen in GdnHCl, and the initial contraction in urea too appears to be gradual.

Mutational Studies Uncover Non-Native Structure in the Dimeric Kinetic Intermediate of the H2A–H2B Heterodimer

The folding pathway of the histone H2A–H2B heterodimer minimally includes an on-pathway, dimeric, burst-phase intermediate, I2. The partially folded H2A and H2B monomers populated at equilibrium were characterized as potential monomeric kinetic intermediates. Folding kinetics were compared for initiation from isolated, folded monomers and the heterodimer unfolded in 4 M urea. The observed rates were virtually identical above 0.4 M urea, exhibiting a log-linear relationship on the final denaturant concentration. Below ∼0.4 M urea (concentrations inaccessible from the 4-M urea unfolded state), a rollover in the rates was observed; this suggests that a component of the I2 ensemble contains non-native structure that rearranges/isomerizes to a more native-like species. The contribution of helix propensity to the stability of the I2 ensemble was assessed with a set of H2A–H2B mutants containing Ala and Gly replacements at nine sites, focusing mainly on the long, central α2 helix. Equilibrium and kinetic folding/unfolding data were collected to determine the effects of the mutations on the stability of I2 and the transition state between I2 and N2. This limited mutational study indicated that residues in the α2 helices of H2A and H2B as well as α1 of H2B and both the C-terminus of α3 and the short αC helix of H2A contribute to the stability of the I2 burst-phase species. Interestingly, at least eight of the nine targeted residues stabilize I2 by interactions that are non-native to some extent. Given that destabilizing I2 and these non-native interactions does not accelerate folding, it is concluded that the native and non-native structures present in the I2 ensemble enable efficient folding of H2A–H2B.

The Unfolded State of the C-Terminal Domain of the Ribosomal Protein L9 Contains Both Native and Non-Native Structure

Interest in the structural and dynamic properties of unfolded proteins has increased in recent years owing to continued interest in protein folding and misfolding. Knowledge of the unfolded state under native conditions is particularly important for obtaining a complete picture of the protein folding process. The C-terminal domain of protein L9 is a globular alpha, beta protein with an unusual mixed parallel and antiparallel beta-strand structure. The folding kinetics and equilibrium unfolding of CTL9 strongly depend on pH, and follow a simple two state model. Both the native and the unfolded state can be significantly populated at pH 3.8 in the absence of denaturant, allowing the native state and the unfolded state to be characterized under identical conditions. Backbone (15)N, (13)C, (1)H and side-chain (13)C(beta), (1)H(beta) chemical shifts, amide proton NOEs, and (15)N R(2) relaxation rates were obtained for the two conformational states at pH 3.8. All the data indicate that the pH 3.8 native state is well folded and is similar to the native state at neutral pH. There is significant residual structure in the pH 3.8 unfolded state. The regions corresponding to the two native state alpha-helices show strong preference to populate helical phi and psi angles. The segment that connects alpha-helix 2 and beta-strand 2 has a significant tendency to form non-native alpha-helical structure. Comparison with the pH 2.0 unfolded state and the urea unfolded state indicates that the tendency to adopt both native and non-native helical structure is stronger at pH 3.8, demonstrating that the unfolded state of CTL9 under native-like conditions is more structured. The implications for the folding of CTL9 are discussed.

Comparison of Insertion and Folding of Chaperone-bound Outer Membrane Protein A (OmpA) of E. coli into Phospholipid Bilayers of Various Composition

OmpA spontaneously inserts and folds into lipid bilayers from a urea-unfolded state upon urea-dilution. Previous work demonstrated that urea can be replaced effectively by the periplasmic chaperone Skp when lipopolysaccharide (LPS), a component of the outer membrane, is present [1]. Skp was shown to bind outer membrane proteins with nanomolar affinity and to prevent their aggregation [2].Here we investigated folding of Skp-bound OmpA into lipid bilayers of different headgroup composition and chain-length, both in absence and presence of LPS. For urea-denatured OmpA and in absence of Skp and LPS, kinetics of folding into bilayers containing the negatively charged phosphatidylglycerol showed a lag-phase of up to 30 min for dilauroylphospholipid bilayers (LUVs, 100 nm diameter) prior to folding. Skp inhibited folding and prolonged the lag-phase at basic pH when LPS was absent. In presence of LPS, no lag-phase was observed and folding rates increased dramatically.When similar experiments were performed with bilayers composed of the corresponding dioleolylphospholipids (SUVs), a lag-phase was not observed, but Skp inhibited very strongly. LPS again stimulated OmpA insertion and folding, albeit not as much as observed for dilaurylphospholipid bilayers.Skp inhibited folding also in experiments with neutral phosphatidylcholine bilayers, irrespective of lipid chain-length. Here, LPS could also facilitate folding of Skp-bound OmpA, but the effect was less pronounced for dioleoylphosphatidylcholine bilayers. The data suggest, LPS-assisted folding of Skp-bound OmpA depends on both, the surface charge of the membrane and on the lipid-chain composition.

Backbone and 13C and 1H assignments for acid unfolded state and urea unfolded state of CTL9

Backbone and 13C and 1H assignments for acid unfolded state and urea unfolded state of CTL9

The Low-pH Unfolded State of the C-Terminal Domain of the Ribosomal Protein L9 Contains Significant Secondary Structure in the Absence of Denaturant but Is No More Compact Than the Low-pH Urea Unfolded State

There is considerable interest in the properties of the unfolded states of proteins, particularly unfolded states which can be populated in the absence of high concentrations of denaturants. Interest in the unfolded state ensemble reflects the fact that it is the starting point for protein folding as well as the reference state for protein stability studies and can be the starting state for pathological aggregation. The unfolded state of the C-terminal domain (residues 58-149) of the ribosomal protein L9 (CTL9) can be populated in the absence of denaturant at low pH. CTL9 is a 92-residue globular alpha, beta protein. The low-pH unfolded state contains more secondary structure than the low-pH urea unfolded state, but it is not a molten globule. Backbone ( (1)H, (13)C, and (15)N) NMR assignments as well as side chain (13)C beta and (1)H beta assignments and (15)N R 2 values were obtained for the pH 2.0 unfolded form of CTL9 and for the urea unfolded state at pH 2.5. Analysis of the deviations of the chemical shifts from random coil values indicates that residues that comprise the two helices in the native state show a clear preference for adopting helical phi and psi angles in the pH 2.0 unfolded state. There is a less pronounced but nevertheless clear tendency for residues 107-124 to preferentially populate helical phi and psi values in the unfolded state. The urea unfolded state has no detectable tendency to populate any type of secondary structure even though it is as compact as the pH 2.0 unfolded state. Comparison of the two unfolded forms of CTL9 provides direct experimental evidence that states which differ significantly in their secondary structure can have identical hydrodynamic properties. This in turn demonstrates that global parameters such as R h or R g are very poor indicators of "random coil" behavior.

Influence of local and residual structures on the scaling behavior and dimensions of unfolded proteins

Although recent spectroscopic studies of chemically denatured proteins hint at significant nonrandom residual structure, the results of extensive small angle X-ray scattering studies suggest random coil behavior, calling for a coherent understanding of these seemingly contradicting observations. Here, we report the results of a Monte Carlo study of the effects of two types of local structures, alpha helix and Polyproline II (PPII) helix, on the dimensions of random coil polyalanine chains viewed as a model of highly denatured proteins. We find that although Flory's power law scaling, long regarded as a signature of random coil behavior, holds for chains containing up to 90% alpha or PPII helix, the absolute magnitude of the chain dimensions is sensitive to helix content. As residual alpha helix content increases, the chain contracts until it reaches a minimum radius at approximately 70% helix, after which the chain dimensions expand rapidly. With an alpha helix content of approximately 20%, corresponding to the Ramachandran probability of being in the helical basin, experimentally observed radii of gyration are recovered. Experimental radii are similarly recovered at an alpha helix content of approximately 87%, providing an explanation for the previously puzzling experimental finding that the dimensions of the highly helical methanol-induced unfolded state are experimentally indistinguishable from those of the helix-poor urea-unfolded state. In contrast, the radius of gyration increases monotonically with increasing PPII content, and is always more expanded than the dimensions observed experimentally. These results suggest that PPII is unlikely the sole, dominant preferred conformation for unfolded proteins.

Chloride-ion concentration dependence of molecular dimension in the acid-denatured state of equine β-lactoglobulin

The chloride-ion concentration dependence of the molecular dimension in the acid-denatured state of equine β-lactoglobulin (ELG) was investigated by small-angle X-ray scattering. In the presence of chloride ion, ELG has a globular and compact conformation (the A state). The molecular dimension of ELG increases little with decreasing chloride-ion concentration. A remarkable dependence was observed for a mutant protein in which both Cys66 and Cys160 were replaced with Ala (C66A/C160A). In the presence of chloride ion, C66A/C160A has a globular and compact conformation, like the wild type. In the absence of chloride ion, however, the molecular dimension and shape was close to that in the urea-unfolded state. Previously, we have shown that the helix content in the acid-denatured state increases with decreasing chloride-ion concentration [Yamada et al. (2006). Proteins Struct. Funct. Bioinf. 63, 595–602]. These results suggest that the secondary structure in the A state is mainly determined by non-local interactions. When they are absent in an expanded conformation, the local interactions become predominant and the amount of non-native α-helix increases.

Native and nonnative conformational preferences in the urea-unfolded state of barstar.

The refolding of barstar from its urea-unfolded state has been studied extensively using various spectroscopic probes and real-time NMR, which provide global and residue-specific information, respectively, about the folding process. Here, a preliminary structural characterization by NMR of barstar in 8 M urea has been carried out at pH 6.5 and 25 degrees C. Complete backbone resonance assignments of the urea-unfolded protein were obtained using the recently developed three-dimensional NMR techniques of HNN and HN(C)N. The conformational propensities of the polypeptide backbone in the presence of 8 M urea have been estimated by examining deviations of secondary chemical shifts from random coil values. For some residues that belong to helices in native barstar, 13C(alpha) and 13CO secondary shifts show positive deviations in the urea-unfolded state, indicating that these residues have propensities toward helical conformations. These residues are, however, juxtaposed by residues that display negative deviations indicative of propensities toward extended conformations. Thus, segments that are helical in native barstar are unlikely to preferentially populate the helical conformation in the unfolded state. Similarly, residues belonging to beta-strands 1 and 2 of native barstar do not appear to show any conformational preferences in the unfolded state. On the other hand, residues belonging to the beta-strand 3 segment show weak nonnative helical conformational preferences in the unfolded state, indicating that this segment may possess a weak preference for populating a helical conformation in the unfolded state.

Glutamine 53 is a gatekeeper residue in the FK506 binding protein.

The effect of non-random conformational averaging in the urea-unfolded state on the folding pathway has been investigated in a variant of the FK506 binding protein with three additional residues at the amino terminus (FKBP(*)). Three mutations (asparagine, aspartate, and threonine) were introduced into position Q53 to enhance formation of non-native helix observed in this part of the protein in the urea-unfolded state. NMR analysis showed minor structural changes in the native state of each mutant, but additional medium-range alphaN(i,i+2) of each mutant nuclear Overhauser enhancements were observed in the urea-unfolded state that were not in FKBP(*), indicating that the mutations had a more substantial effect on the unfolded state ensemble than on the native state ensemble. Isothermal equilibrium denaturation measurements showed that the Q53T and Q53D mutants were destabilized, whereas the Q53N mutant was stabilized relative to FKBP(*) with little change in the equilibrium m values. The unfolding rates of Q53N and Q53T were similar to that of FKBP(*), but Q53D unfolded twice as fast as FKBP(*). In contrast, the mutations had a more pronounced effect on the refolding kinetics. Q53N refolded slightly faster and exhibited a kinetic folding intermediate similar to that of FKBP(*). The Q53D and Q53T mutants also refolded faster than FKBP(*) but lacked the folding intermediate, indicating that these mutants experienced a different folding trajectory and transition state than FKBP(*) and Q53N. The refolding kinetic Phi values were 0.74, 1.4 and 7.9 for Q53N, Q53T, and Q53D, respectively. The data point to Q53 functioning as a gatekeeper residue in the folding of FKBP(*). This study shows that perturbing the unfolded state ensemble via mutagenesis can provide insights into residues that play important roles in the folding pathway, and represents an attractive strategy for mapping the high-energy portions of the folding energy landscape.

Probing the equilibrium denaturation of the serpin α1-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state

The native conformation of proteins in the serpin superfamily is metastable. In order to understand why serpins attain the native state instead of more stable conformations we have begun investigations into the equilibrium-unfolding of α1-antitrypsin. α1-Antitrypsin contains two tryptophan residues, Trp194 and Trp238, situated on the A and B β-sheets, respectively. Site-directed mutagenesis was used to construct two single-tryptophan variants. Both variants were fully active and had similar secondary structure and stabilities to α1-antitrypsin. The denaturation of α1-antitrypsin and its variants was extremely similar when followed by far-UV CD, indicating the presence of a single intermediate. Fluorescence analysis of the unfolding behavior of each single tryptophan variant indicated that the sole tryptophan residue reported the structural changes within its immediate environment. These data suggest that the A β-sheet is expanded in the intermediate state whilst no structural change around the B β-sheet has occurred. In the urea-induced unfolded state, Trp238 does not become fully solvated, suggesting the persistence of structure around this residue. The implications of these data on the folding, misfolding and function of the serpin superfamily are discussed.

Characterization of a Partially Folded Monomer of the DNA-binding Domain of Human Papillomavirus E2 Protein Obtained at High Pressure

The pressure-induced dissociation of the dimeric DNA binding domain of the E2 protein of human papillomavirus (E2-DBD) is a reversible process with a Kd of 5.6 x 10(-8) M at pH 5.5. The complete exposure of the intersubunit tryptophans to water, together with the concentration dependence of the pressure effect, is indicative of dissociation. Dissociation is accompanied by a decrease in volume of 76 ml/mol, which corresponds to an estimated increase in solvent-exposed area of 2775 A2. There is a decrease in fluorescence polarization of tryptophan overlapping the red shift of fluorescence emission, supporting the idea that dissociation of E2-DBD occurs in parallel with major changes in the tertiary structure. The dimer binds bis(8-anilinonaphthalene-1-sulfonate), and pressure reduces the binding by about 30%, in contrast with the almost complete loss of dye binding in the urea-unfolded state. These results strongly suggest the persistence of substantial residual structure in the high pressure state. Further unfolding of the high pressure state was produced by low concentrations of urea, as evidenced by the complete loss of bis(8-anilinonaphthalene-1-sulfonate) binding with less than 1 M urea. Following pressure dissociation, a partially folded state is also apparent from the distribution of excited state lifetimes of tryptophan. The combined data show that the tryptophans of the protein in the pressure-dissociated state are exposed long enough to undergo solvent relaxation, but the persistence of structure is evident from the observed internal quenching, which is absent in the completely unfolded state. The average rotational relaxation time (derived from polarization and lifetime data) of the pressure-induced monomer is shorter than the urea-denatured state, suggesting that the species obtained under pressure are more compact than that unfolded by urea.

The native and the heat-induced denatured states of α-chymotrypsinogen A: thermodynamic and spectroscopic studies

We report the first protein phase-diagram characterized by a combination of volumetric, calorimetric, and spectroscopic techniques. More specifically, we use ultrasonic velocimetry, densimetry, and differential scanning calorimetry, in conjunction with UV absorbance and CD spectroscopy to detect and to characterize the conformational transitions of α-chymotrypsinogen A as a function of both pH and temperature. As judged by the CD spectra, we find that, at room temperature, the protein remains in the native state over the entire pH range investigated (pH 1 to 10). The melting profiles of the native state reveal three distinct pH domains in which protein denaturation produces different final states. Below pH 3.1, we find the heat-induced denatured state of the protein to be molten globule (MG), lacking the native-like tertiary structure, while exhibiting significant secondary structural elements. At neutral and alkaline pH, we find the heat-induced denatured state to be unfolded (U), lacking both tertiary and secondary structures, while being structurally similar to the urea-unfolded state. At intermediate pH values (between pH 3.1 and 7), we find the heat-induced denatured state to exhibit properties characteristic of both the MG and U states. Although at room temperature the protein remains native within the whole pH range studied (pH 1 to 10), our volumetric data reveal that the native state slightly “softens” at low pH, probably, due to pH-induced alterations in electrostatic forces causing the packing of the protein interior at low pH and room temperature to become less “tight”. This softening of the protein at low pH is reflected in an 8% increase in the intrinsic compressibility, k M, of the protein “native” state. Our volumetric data also allow us to conclude that the heat-induced MG state retains a liquid-like, water-inaccessible core, with a volume that corresponds to about 40% of the solvent-inaccessible core of the native state. By contrast, our volumetric data are consistent with the U state of the protein being essentially unfolded, with the majority of its constituent atomic groups being solvent exposed and, therefore, strongly hydrated.

Urea-Induced Equilibrium Unfolding of Single Tryptophan Mutants of Yeast Phosphoglycerate Kinase: Evidence for a Stable Intermediate

Several single-tryptophan mutants of yeast phosphoglycerate kinase (PGK) have been used in the present study to characterize the urea-induced unfolding of PGK. A possibility that residual structures might be present in the urea-unfolded state was also investigated. The urea-induced unfolding transitions were monitored using circular dichroism (CD) and fluorescence techniques. The presence of stable intermediate(s) during urea-induced unfolding is suggested by biphasic transitions detected for the mutants containing tryptophans in the N-terminal domain and by the noncoincidence of transitions detected by various methods for other mutants. The N-terminal tryptophan probes exhibit hyperfluorescent properties in the intermediate state and a wavelength of maximum emission that lies between that of the native and unfolded state. This unfolding intermediate exhibits a major decrease in the ellipticity at 220 nm, but only a minor decrease at 278 nm, relative to the native state. These results suggest a significant loss of secondary structure content and a relatively small change in the asymmetric environment of tyrosine residues. Increased 1-anilinonaphthalene-8-sulfonic acid binding in the denaturant concentration range corresponding to the N → I transition indicates the presence of a partially folded structure with exposed hydrophobic surfaces. These results demonstrate that the partially folded intermediates detected during urea-induced denaturation are structurally similar to those detected previously during guanidine-induced denaturation. No significant differences were detected between the urea- and guanidine-unfolded proteins on the basis of their fluorescence and CD properties.

Evaluating the effects of a single amino acid substitution on both the native and denatured states of a protein.

For proteins that contain a disulfide bond, stability is linked thermodynamically to thiol-disulfide exchange. We use this relationship to obtain unfolding free energies for both the reduced and oxidized forms of Escherichia coli thioredoxin from measurements of the effective concentrations of protein thiols. We then evaluate the effect of an amino acid substitution on disulfide bond formation in both the native and denatured states of the protein. Although the Pro-34----Ser substitution in thioredoxin results in a decrease of the effective concentration of protein thiols in the native state, the effective concentration increases in the denatured state. The net effect of the amino acid substitution is to increase the stability of reduced thioredoxin by approximately 2.4 kcal/mol, whereas the stability of the oxidized protein remains the same. By assuming a two-state unfolding equilibrium and a mutation free energy of -7.7 kcal/mol for the Pro-34----Ser substitution in the reduced, urea-unfolded state (based on estimates of solvation and entropic changes), we obtained relative free energies for the native and denatured states of the mutant and wild-type proteins, in both the reduced and oxidized forms.