Chemical denaturation: potential impact of undetected intermediates in the free energy of unfolding and m-values obtained from a two-state assumption.

The chemical unfolding transition of a protein was simulated, including the presence of an intermediate (I) in equilibrium with the native (N) and unfolded (U) states. The calculations included free energies of unfolding, DeltaGuw, in the range of 1.4 kcal/mol to 10 kcal/mol and three different global m-values. The simulations included a broad range of equilibrium constants for the N left arrow over right arrow I process. The dependence of the N <--> I equilibrium on the concentration of denaturant was also included in the simulations. Apparent DeltaGuw and m-values were obtained from the simulated unfolding transitions by fitting the data to a two-state unfolding process. The potential errors were calculated for two typical experimental situations: 1) the unfolding is monitored by a physical property that does not distinguish between native and intermediate states (case I), and 2) the physical property does not distinguish between intermediate and unfolded states (case II). The results obtained indicated that in the presence of an intermediate, and in both experimental situations, the free energy of unfolding and the m-values could be largely underestimated. The errors in DeltaGuw and m-values do not depend on the m-values that characterize the global N <--> U transition. They are dependent on the equilibrium constant for the N <--> I transition and its characteristic m1-value. The extent of the underestimation increases for higher energies of unfolding. Including no random error in the simulations, it was estimated that the underestimation in DeltaGuw could range between 25% and 35% for unfolding transitions of 3-10 kcal/mol (case I). In case II, the underestimation in DeltaGuw could be even larger than in case I. In the same energy range, a 50% error in the m-value could also take place. The fact that most of the mutant proteins are characterized by both a lower m-value and a lower stability than the wild-type protein suggests that in some cases the results could have been underestimated due to the application of the two-state assumption.     

Rapid unfolding of a domain populates an aggregation-prone intermediate that can be recognized by GroEL

Some amino acid substitutions in phage P22 coat protein cause a temperature-sensitive folding (tsf) phenotype. In vivo, these tsf amino acid substitutions cause coat protein to aggregate and form intracellular inclusion bodies when folded at high temperatures, but at low temperatures the proteins fold properly. Here the effects of tsf amino acid substitutions on folding and unfolding kinetics and the stability of coat protein in vitro have been investigated to determine how the substitutions change the ability of coat protein to fold properly. The equilibrium unfolding transitions of the tsf variants were best fit to a three-state model, N if I if U, where all species concerned were monomeric, a result confirmed by velocity sedimentation analytical ultracentrifugation. The primary effect of the tsf amino acid substitutions on the equilibrium unfolding pathway was to decrease the stability (DeltaG) and the solvent accessibility (m-value) of the N if I transition. The kinetics of folding and unfolding of the tsf coat proteins were investigated using tryptophan fluorescence and circular dichroism (CD) at 222 nm. The tsf amino acid substitutions increased the rate of unfolding by 8-14-fold, with little effect on the rate of folding, when monitored by tryptophan fluorescence. In contrast, when folding or unfolding reactions were monitored by CD, the reactions were too fast to be observed. The tsf coat proteins are natural substrates for the molecular chaperones, GroEL/S. When native tsf coat protein monomers were incubated with GroEL, they bound efficiently, indicating that a folding intermediate was significantly populated even without denaturant. Thus, the tsf coat proteins aggregate in vivo because of an increased propensity to populate this unfolding intermediate.     

Conformational plasticity in folding of the split beta-alpha-beta protein S6: evidence for burst-phase disruption of the native state

An increasing number of folding studies of two-state proteins shows that point mutations sometimes change the kinetic m-values, leading to kinks and curves in the chevron plots. The molecular origin of these changes is yet unclear although it is speculated that they are linked to structural rearrangement of the transition state or to accumulation of meta-stable intermediates. To shed more light on this issue, we present here a combined m and phi-value analysis of the split beta-alpha-beta protein S6. Wild-type S6 displays classical two-state kinetics with v-shaped chevron plot, but a majority of its mutants display distinct m-value changes or curved chevrons. We observe that this kinetic aberration of S6 is linked to mutations that are clustered in distinct regions of the native structure. The most pronounced changes, i.e. decrease in the m-value for the unfolding rate constant, are seen upon truncation of interactions between the N and C termini, whereas mutations in the centre of the hydrophobic core show smaller or even opposed effects. As a consequence, the calculated phi-values display a systematic increase upon addition of denaturant. In the case of S6, the phenomenon seems to arise from a general plasticity of the different species on the folding pathway. That is, the structure of the denatured ensemble, the transition state, and the native ground-state for unfolding seem to change upon mutation. From these changes, it is concluded that interactions spanning the centre of the hydrophobic core form early in folding, whereas the entropically disfavoured interactions linking the N and C termini consolidate very late, mainly on the down-hill-side of the folding barrier.     

The magnitude of changes in guanidine-HCl unfolding m-values in the protein, iso-1-cytochrome c, depends upon the substructure containing the mutation

Hydrophilic to hydrophobic mutations have been made at 11 solvent exposed sites on the surface of iso-1-cytochrome c. Most of these mutations involve the replacement of lysine with methionine, which is nearly isosteric with lysine. Minimal perturbation to the native structure is expected, and this expectation is confirmed by infrared amide I spectroscopy. Guanidine hydrochloride denaturation studies demonstrate that these variants affect the magnitude of the m-value, the rate of change of free energy with respect to denaturant concentration, to different degrees. Changes in m-values are indicative of changes in the equilibrium folding mechanism of a protein. Decreases in m-values are normally thought to result either from an increased population of intermediates during unfolding or from a more compact denatured state. When cytochrome c is considered in terms of its thermodynamic substructures, the changes in the m-value for a given variant appear to depend upon the substructure in which the mutation is made. These data indicate that the relative stabilities and physical properties of substructures of cytochrome c play an important determining role in the equilibrium folding mechanism of this protein.     

Detection and characterization of partially unfolded oligomers of the SH3 domain of alpha-spectrin

For the purpose of equilibrium and kinetic folding-unfolding studies, the SH3 domain of alpha-spectrin (spc-SH3) has long been considered a classic two-state folding protein. In this work we have indeed observed that the thermal unfolding curves of spc-SH3 measured at pH 3.0 by differential scanning calorimetry, circular dichroism, and NMR follow apparently the two-state model when each unfolding profile is considered individually. Nevertheless, we have found that protein concentration has a marked effect upon the thermal unfolding profiles. This effect cannot be properly explained in terms of the two-state unfolding model and can only be interpreted in terms of the accumulation of intermediate associated states in equilibrium with the monomeric native and unfolded states. By chemical cross-linking and pulsed-field gradient NMR diffusion experiments we have been able to confirm the existence of associated states formed during spc-SH3 unfolding. A three-state model, in which a dimeric intermediate state is assumed to be significantly populated, provides the simplest interpretation of the whole set of thermal unfolding data and affords a satisfactory explanation for the concentration effects observed. Whereas at low concentrations the population of the associated intermediate state is negligible and the unfolding process consequently takes place in a two-state fashion, at concentrations above approximately 0.5 mM the population of the intermediate state becomes significant at temperatures between 45 degrees C and 80 degrees C and reaches up to 50% at the largest concentration investigated. The thermodynamic properties of the intermediate state implied by this analysis fall in between those of the unfolded state and the native ones, indicating a considerably disordered conformation, which appears to be stabilized by oligomerization.     

Interleukin-1 beta folding between pH 5 and 7: experimental evidence for three-state folding behavior and robust transition state positions late in folding

The thermodynamic stability and folding kinetics of the all beta-sheet protein interleukin-1beta were measured between 0 and 4 M GdmCl concentrations and pH 5-7. Native interleukin-1beta undergoes a 3.5 kcal/mol decrease in thermodynamic stability, Delta, as pH is increased from 5 to 7. The native state parameter m(NU), measuring protein destabilization/[GdmCl], remains constant between pH 5 and 7, indicating that the solvent-exposed surface area difference between the native state and unfolded ensemble is unchanged across this pH range. Similarly, pH changes between 5 and 7 decrease only the thermodynamic stability, DeltaG(H)2(O), and not the m-values, of the kinetic intermediate and transition states. This finding is shown to be consistent with transition state configurations which continue to be the high-energy configurations of the transition state in the face of changing stability conditions. A three-state folding mechanism U right arrow over left arrow I right arrow over left arrow N is shown to be sufficient in characterizing IL-1beta folding under all conditions studied. The m-values of refolding transitions are much larger than the m-values of unfolding transitions, indicating that that the fast, T(2) (U right arrow over left arrow I), and slow, T(1) (I right arrow over left arrow N), transition states are highly similar to the intermediate I and native state N, respectively. Many of the folding properties of interleukin-1beta are shared among other members of the beta-trefoil protein family, although clear differences can exist.     

Local/bulk determinants of conformational stability of exchangeable apolipoproteins

GuHCl-induced denaturation of human plasma apoA-I, apoA-II, apoA-IV, apoE3 and three recombinant apoE isoforms in solution and discoidal complexes with phosphatidylcholine (only plasma proteins) was studied. The protein conformational stability (ΔG(H(2)O)) and a slope of linear dependence of free energy of unfolding on GuHCl concentration (m-value) were estimated with the three equilibrium schemes. The data for all proteins, except apoA-II, fit with the three-state model, thus evidencing two-domain structure. The predicted folding rate of the four apoE in solution correlated with conformational stability. The dependence disappeared at the inclusion of apoA-I and apoA-IV into analysis and the m-values, adjusted for residue number in helices (m(rh)), differed between those for apoE and apoA-I/apoA-IV. However, the m(rh)-values for six proteins correlated positively with the fractional change in accessible surface area at unfolding for Phe, Lys and Asn, while negatively for Arg, Ala and Gly residues. The difference between the adjusted ΔG(rh)(H(2)O) values for apolipoproteins in complexes and in solution decreased at the increase of reduced temperature (T(obs)-T(t))/T(t). The induction of intrinsic disorder by arginine residues may be of primary importance in metabolism and function of exchangeable apolipoproteins, while their stability in nascent discoidal HDL is controlled by the physical state of phosphatidylcholine.     

Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9

The kinetics and thermodynamics of the folding of the homologous four-helix proteins Im7 and Im9 have been characterised at pH 7.0 and 10 degrees C. These proteins are 60 % identical in sequence and have the same three-dimensional structure, yet appear to fold by different kinetic mechanisms. The logarithm of the folding and unfolding rates of Im9 change linearly as a function of urea concentration and fit well to an equation describing a two-state mechanism (with a folding rate of 1500 s-1, an unfolding rate of 0. 01 s-1, and a highly compact transition state that has approximately 95 % of the native surface area buried). By contrast, there is clear evidence for the population of an intermediate during the refolding of Im7, as indicated by a change in the urea dependence of the folding rate and the presence of a significant burst phase amplitude in the refolding kinetics. Under stabilising conditions (0.25 M Na2SO4, pH 7.0 and 10 degrees C) the folding of Im9 remains two-state, whilst under similar conditions (0.4 M Na2SO4, pH 7.0 and 10 degrees C) the intermediate populated during Im7 refolding is significantly stabilised (KUI=125). Equilibrium denaturation experiments, under the conditions used in the kinetic measurements, show that Im7 is significantly less stable than Im9 (DeltaDeltaG 9.3 kJ/mol) and the DeltaG and m values determined accord with those obtained from the fit to the kinetic data. The results show, therefore, that the population of an intermediate in the refolding of the immunity protein structure is defined by the precise amino acid sequence rather than the global stability of the protein. We discuss the possibility that the intermediate of Im7 is populated due to differences in helix propensity in Im7 and Im9 and the relevance of these data to the folding of helical proteins in general.     

Three-state denaturation of alpha-lactalbumin by guanidine hydrochloride.

The reversible unfolding of α-lactalbumin by guanidine hydrochloride has been studied at 25.0 °C by means of ultraviolet circular dichroism measurements. The non-coincidence of the apparent transition curves obtained from the ellipticity changes at far (222 nm) and at near (270 nm and 296 nm) ultraviolet wave-lengths demonstrates the presence of at least one intermediate in the denaturation process. The aromatic residues which contribute to the Cotton effects at 270 nm and at 296 nm appear to be exposed to solvent in the first stage of a two-stage process, while the helical regions of the polypeptide chain appear to be destroyed in the second stage. Earlier work has demonstrated an acid transition between two compact forms of α-lactalbumin, a native (neutral pH) form and an acid form. Results presented here suggest that the acid form is produced as an intermediate in the first stage of total unfolding at neutral pH.Lysozyme and α-lactalbumin are known to have similar primary structures and are expected to have similar tertiary structures, but several differences in their properties have been described. The comparison of the unfolding transitions of α-lactalbumin and lysozyme provides a result compatible with similar tertiary structures, although the free energy of stabilization of the native state is 3 to 5 kcal/mol smaller for α-lactalbumin than for lysozyme. The pH dependence of the unfolding reaction can be described in terms of abnormal histidyl and carboxyl residues. The presence of a stable intermediate in the denaturation process may cause a difference in dynamic character in the native state between the two proteins and thus provide a reasonable interpretation for their known differences in chemical reactivity.     

Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: spectroscopic description of the native, intermediate, and unfolded states

Ervatamin A, a cysteine proteases from Ervatamia coronaria, has been used as model system to examine structure-function relationship by equilibrium unfolding methods. Ervatamin A belongs to alpha+beta class of proteins and exhibit stability towards temperature and chemical denaturants. Acid induced unfolding of ervatamin A was incomplete with respect to the structural content of the enzyme. Between pH 0.5 and 2.0, the enzyme is predominantly in beta-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (I(A) state). Surprisingly, high concentrations of GuHCl required to unfold this state and the transition mid points GuHCl induced unfolding curves are significantly higher. GuHCl induced unfolding of ervatamin A at pH 3.0 as well as at pH 4.0 is complex and cannot be satisfactorily fit to a two-state model for unfolding. Besides, a strong ANS binding to the protein is observed at low concentration of GuHCl, indicating the presence of intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8M) the enzyme retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to urea unfolding at pH 3.0 and below. Urea induced unfolding of ervatamin A at pH 3.0 is cooperative and the transitions curves obtained by different probes are and non-coincidental. Temperature denaturation of ervatamin A in I(A) state is non-cooperative, contrary to the cooperativity seen with native protein, suggesting the presence of two parts in the molecular structure of ervatamin A may be domains, with different stability that unfolds in steps. Careful inspection of biophysical properties of intermediate states populated in urea and GuHCl (I(UG) state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (I(A) state) identified at pH approximately 1.5 are different from those of the I(UG) state.     

Folding/Unfolding Kinetics of Apomyoglobin

The equilibrium and kinetic folding/unfolding of apomyoglobin (ApoMb) were studied at pH 6.2, 11 °C by recording tryptophan fluorescence. The equilibrium unfolding of ApoMb in the presence of urea was shown to involve accumulation of an intermediate state, which had a higher fluorescence intensity as compared with the native and unfolded states. The folding proceeded through two kinetic phases, a rapid transition from the unfolded to the intermediate state and a slow transition from the intermediate to the native state. The accumulation of the kinetic intermediate state was observed in a wide range of urea concentrations. The intermediate was detected even in the region corresponding to the unfolding limb of the chevron plot. Urea concentration dependence was obtained for the observed folding/unfolding rate. The shape of the dependence was compared with that of two-state proteins characterized by a direct transition from the unfolded to the native state.     

HX-ESI-MS and optical studies of the unfolding of thioredoxin indicate stabilization of a partially unfolded, aggregation-competent intermediate at low pH

Hydrogen exchange monitored by mass spectrometry (HX-MS), in conjunction with multiple optical probes, has been used to characterize the unfolding of thioredoxin. Equilibrium and kinetic studies have been carried out at pH 7 and 3. The HX-MS measurements are shown to be capable of distinguishing between native (N) and unfolded (U) protein molecules when both are present together, and their application in kinetic experiments allows the unfolding reaction to be delineated from the proline isomerization reaction to which it is coupled. At pH 7, equilibrium unfolding studies monitored by three optical probes, intrinsic fluorescence at 368 nm, ellipticity at 222 nm, and ellipticity at 270 nm, as well as by HX-MS, indicate that no intermediate is populated at pH 7, the unfolding reaction is slower than the proline isomerization reaction that follows it, and the three optical probes yield identical kinetics for unfolding, which occurs in a single kinetic phase. The fractional change in any of the three optical signals at any time of unfolding predicts the fraction of the molecules that have become U, as determined by HX-MS. Hence, unfolding at pH 7 appears to occur via a two-state N <==> U mechanism. In contrast at pH 3, HX-MS as well as optical measurements indicate that an unfolding intermediate is stabilized and hence accumulates in equilibrium with N and U, at concentrations of denaturant that define the transition zone of the equilibrium unfolding curve. The intermediate has lost the near-UV signal characteristic of N and possesses fewer amide hydrogen sites that are stable to exchange than does N. Kinetic experiments at pH 3, where unfolding is much faster than proline isomerization, show that more than one intermediate accumulates transiently during unfolding. Thus, the unfolding of thioredoxin occurs via an N <==> I <==> U mechanism, where I is a partially unfolded intermediate that is stabilized and hence populated at pH 3 but not at pH 7. It is shown that transient aggregation of this intermediate results in a deceleration of the kinetics of unfolding at high protein concentrations at pH 3 but not at pH 7.     

Partially unfolded equilibrium state of hen lysozyme studied by circular dichroism spectroscopy

Equilibrium unfolding of hen egg white lysozyme as a function of GdnCl concentration at pH 0.9 was studied over a temperature range 268.2-303.2 K by means of CD spectroscopy. As monitored by far- and near-UV CD at 222 and 289 nm, the lack of coincidence between two unfolding transition curves was observed, which suggests the existence of a third conformational species in addition to native and unfolded states. The three-state model, in which a stable intermediate is populated, was employed to estimate the thermodynamic parameters for the GdnCl-induced unfolding. It was found that the transition from the native to intermediate states proceeds with significant changes in enthalpy and entropy due to an extremely cooperative process, while the transition from the intermediate to unfolded states shows a low cooperativity with small enthalpy and entropy changes. These results indicate that the highest energy barrier for the GdnCl-induced unfolding of hen lysozyme is located in the process from the native state to the intermediate state, and this process is largely responsible for the cooperativity of protein unfolding.     

Stability and cooperativity of individual tertiary contacts in RNA revealed through chemical denaturation

For proteins, understanding tertiary interactions involved in local versus global unfolding has become increasingly important for understanding the nature of the native state ensemble, the mechanisms of unfolding, and the stability of both the native and intermediate states in folding. In this work we have addressed related questions with respect to RNA structure by combining chemical denaturation and hydroxyl radical footprinting methods. We have determined unfolding isotherms for each of 26 discrete sites of protection located throughout the Tetrahymena thermophila group I ribozyme. The cooperativity of folding, m-value, and the free energy, DeltaG degrees N-U, associated with formation of each tertiary contact was determined by analysis of the isotherms. The DeltaG degrees N-U values measured in this study vary from 1.7 +/- 0.2 to 7. 6 +/- 1.2 kcal mol-1. Thus, the stability of these discrete tertiary contacts vary by almost 104. In addition, an intradomain contact and three interdomain contacts show high cooperativity (m-values of 1.1 +/- 0.2 to 1.7 +/- 0.3 kcal mol-1 M-1) indicating that these contacts exhibit global cooperatively in their folding behavior. This new approach to examining RNA stability provides an exciting comparison to our understanding of protein structure and folding mechanisms.     

Analysis of the stability of multimeric proteins by effective DeltaG and effective m-values

Analyzing the stability of a multimeric protein is challenging because of the intrinsic difficulty in handling the mathematical model for the folded multimer-unfolded monomer equilibrium. To circumvent this problem, we introduce the concept of effective stability, DeltaGeff (= -RTlnKeff), where Keff is the equilibrium constant expressed in monomer units. Analysis of the denaturant effect on DeltaGeff gives new insight into the stability of multimeric proteins. When a multimeric protein is mostly folded, the dependence of effective stability on denaturant concentration (effective m-value) is simply the m-value of its monomeric unit. However, when the protein is mostly unfolded, its stability depends on denaturant concentration with the m-value of its multimeric form. We also find that the effective m-value at the Cm is a good approximation of the apparent m-value determined by fitting the equilibrium unfolding data from multimeric proteins with a two-state monomer model. Moreover, when the m-value of a monomeric unit is estimated from its size, the effective stability of a multimeric protein can be determined simply from Cm and this estimated m-value. These simple and intuitive approaches will allow a facile analysis of the stability of multimeric proteins. These analyses are also applicable for high-throughput analysis of protein stability on a proteomic scale.     

Acid-induced equilibrium folding intermediate of human platelet profilin

The acid-induced unfolding of human platelet profilin (HPP) can be minimally modeled as a three-state process. Equilibrium unfolding studies have been performed on human platelet profilin1 (HPP) and monitored by far-UV circular dichroism, tryptophan fluorescence, ANS binding, and NMR spectroscopy. Far-UV CD measurements obtained by acid titration demonstrate that HPP unfolds via a three-state mechanism (N --> I --> U), with a highly populated intermediate between pH 4 and 5. Approximately 80% of native helical secondary structural content remains at pH 4, as indicated by monitoring the CD signal at 222 nm. The stability (DeltaGH2O) of the native conformation at pH 7.0 (obtained by monitoring the change in tryptophan signal as a function of urea concentration) is 5.56 +/- 0.51 kcal mol-1; however, the DeltaGH2O for the intermediate species at pH 4 is 2.01 +/- 0.47 kcal mol-1. The calculated m-values for the pH 7.0 and pH 4.0 species were 1.64 +/- 0.15 and 1.34 +/- 0.17 kcal mol-1 M-1, respectively, which is an indication that the native and intermediate species are similarly compact. Additionally, translational diffusion measurements obtained by NMR spectroscopy and ANS binding studies are consistent with a globular and compact conformation at both pH 7.0 and 4.0. The pKa values for the two histidine (His) residues located on helix 4 of HPP were determined to be 5.6 and 5.7 pH units. These pKa values coincide with the midpoint of the far-UV CD acid titration curve and suggest that the protonation of one or both His residues may play a role in the formation of the unfolding intermediate. Stable intermediate species populate the 2D 1H-15N HSQC NMR spectra between pH 4 and 5. A number of backbone and side-chain resonances show significant perturbations relative to the native spectrum; however, considerable nativelike tertiary contacts remain. Interestingly, the residues on HPP that are significantly altered at low pH coincide with segments of the G-actin binding surface and poly-l-proline binding interface. The earlier reports that a decrease in pH below 6.0 induces structural alterations in profilin, favoring dissociation of the profilin-actin complex, corresponds with the structural alterations observed in the partially unfolded species. Our findings suggest that a novel mechanism for pH induced disruption of the profilin-G-actin complex involve a nativelike unfolding intermediate of profilin.     

Correspondence between anomalous m- and DeltaCp-values in protein folding

Proteins folding according to a classical two-state system characteristically show V-shaped chevron plots. We have previously interpreted the symmetrically curved chevron plot of the protein U1A as denaturant-dependent movements in the position of the transition state ensemble (TSE). S6, a structural analog of U1A, shows a classical V-shaped chevron plot indicative of straightforward two-state kinetics, but the mutant LA30 has a curved unfolding limb, which is most consistent with TSE mobility. The kinetic m-values (derivatives of the rate constants with respect to denaturant concentration) in themselves depend on denaturant concentration. To obtain complementary information about putative mobile TSEs, we have carried out a thermodynamic analysis of the three proteins, based on data for refolding and unfolding over the range 10 degrees C to 70 degrees C. The data at all temperatures can be fitted to two-state model systems. Importantly, for all three proteins the activation heat capacities are, within error, identical to the heat capacities measured in independent experiments under equilibrium conditions. Although the equilibrium heat capacities are essentially invariant with regard to denaturant concentration, the activation heat capacities, similar to the structurally equivalent kinetic m-values, show marked denaturant dependence. Furthermore, the values of beta++ at different denaturant concentrations measured by m-values and by heat capacity values are very similar. These observations are consistent with significant transition state movements within the framework of two-state folding. The basis for TSE movement appears to be enthalpic rather than entropic, suggesting that the binding energy of denaturant-protein interactions is a major determinant of the response of energy landscape contours to changing environments.     

Investigation of folding/unfolding kinetics of apomyoglobin

Apomyoglobin kinetic and equilibrium unfolding and folding processes were studied at pH 6.2, 11 degrees C by stopped-flow tryptophan fluorescence. There are two distinct consecutive processes in apomyoglobin folding process, namely, the protein fast transition between the unfolded (U) and an intermediate (I) states (U <----> I) and slow transition between the intermediate and the native (N) states (I <----> N). Accumulation of the intermediate state was observed in the wide range of urea concentrations. The presence of the intermediate state was shown even beyond the middle transition on the unfolding limb. The dependence of observed folding/unfolding rates on urea concentration (chevron plot) was obtained. The shape of this dependence was compared with that of two-state proteins, folding from the U to N state.     

Conformational plasticity of cryptolepain: accumulation of partially unfolded states in denaturants induced equilibrium unfolding

pH and chemical denaturant dependent conformational changes of a serine protease cryptolepain from Cryptolepis buchanani are presented in this paper. Activity measurements, near UV, far UV CD, fluorescence emission spectroscopy, and ANS binding studies have been carried out to understand the folding mechanism of the protein in the presence of denaturants. pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. The preliminary biophysical study on cryptolepain shows that major elements of secondary structure are beta-sheets. Under neutral conditions the enzyme was stable in urea while GuHCl-induced equilibrium unfolding was cooperative. Cryptolepain shows little ANS binding even under neutral conditions due to more hydrophobicity of beta-sheets. Multiple intermediates were populated during the pH-induced unfolding of cryptolepain. Temperature-induced denaturation of cryptolepain in the molten globule like state is non-cooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts, possibly domains, in the molecular structure of cryptolepain, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of A state (molten globule state) of cryptolepain is unique, as lower concentration of denaturant, not only induces structure but also facilitate transition from one molten globule like state (MG(1)) into another (MG(2)). The increase of pH drives the protein into alkaline denatured state characterized by the absence of any ANS binding. GuHCl- and urea-induced unfolding transition curves at pH 12.0 were non-coincidental indicating the presence of an intermediate in the unfolding pathway.