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.     

Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase

The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease.

High-sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117-->Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2-state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the beta subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0-100 degrees C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure-based thermodynamic calculations also predict that the most probable intermediate is one in which the beta subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.     

On the role of some conserved and nonconserved amino acid residues in the transitional state and intermediate of apomyoglobin folding

The contributions of some amino acid residues in the A, B, G, and H helices to the formation of the folding nucleus and folding intermediate of apomyoglobin were estimated. The effects of point substitutions of Ala for hydrophobic amino acid residues on the structural stability of the native (N) protein and its folding intermediate (I), as well as on the folding/unfolding rates for four mutant apomyoglobin forms, were studied. The equilibrium and kinetic studies of the folding/unfolding rates of these mutant proteins in a wide range of urea concentrations demonstrated that their native state was considerably destabilized as compared with the wild-type protein, whereas the stability of the intermediate state changed moderately. It was shown that the amino acid residues in the A, G, and H helices contributed insignificantly to the stabilization of the apomyoglobin folding nucleus in the rate-limiting I ? N transition, taking place after the formation of the intermediate, whereas the residue of the B helix was of great importance in the formation of the folding nucleus in this transition.     

Structure of a partially unfolded form of Escherichia coli dihydrofolate reductase provides insight into its folding pathway

Proteins frequently fold via folding intermediates that correspond to local minima on the conformational energy landscape. Probing the structure of the partially unfolded forms in equilibrium under native conditions can provide insight into the properties of folding intermediates. To elucidate the structures of folding intermediates of Escherichia coli dihydrofolate reductase (DHFR), we investigated transient partial unfolding of DHFR under native conditions. We probed the structure of a high‐energy conformation susceptible to proteolysis (cleavable form) using native‐state proteolysis. The free energy for unfolding to the cleavable form is clearly less than that for global unfolding. The dependence of the free energy on urea concentration (m‐value) also confirmed that the cleavable form is a partially unfolded form. By assessing the effect of mutations on the stability of the partially unfolded form, we found that native contacts in a hydrophobic cluster formed by the F‐G and Met‐20 loops on one face of the central β‐sheet are mostly lost in the partially unfolded form. Also, the folded region of the partially unfolded form is likely to have some degree of structural heterogeneity. The structure of the partially unfolded form is fully consistent with spectroscopic properties of the near‐native kinetic intermediate observed in previous folding studies of DHFR. The findings suggest that the last step of the folding of DHFR involves organization in the structure of two large loops, the F‐G and Met‐20 loops, which is coupled with compaction of the rest of the protein.     

Equilibrium and kinetic studies on folding of canine milk lysozyme

Thermal and chemical unfolding studies of the calcium-binding canine lysozyme (CL) by fluorescence and circular dichroism spectroscopy show that, upon unfolding in the absence of calcium ions, a very stable equilibrium intermediate state is formed. At room temperature and pH 7.5, for example, a stable molten globule state is attained in 3 M GdnHCl. The existence of such a pure and stable intermediate state allowed us to extend classical stopped-flow fluorescence measurements that describe the transition from the native to the unfolded form, with kinetic experiments that monitor separately the transition from the unfolded to the intermediate state and from the intermediate to the native state, respectively. The overall refolding kinetics of apo-canine lysozyme are characterized by a significant drop in the fluorescence intensity during the dead time, followed by a monoexponential increase of the fluorescence with k = 3.6 s(-1). Furthermore, the results show that, unlike its drastic effect on the stability, Ca(2+)-binding only marginally affects the refolding kinetics. During the refolding process of apo-CL non-native interactions, comparable to those observed in hen egg white lysozyme, are revealed by a substantial quenching of tryptophan fluorescence. The dissection of the refolding process in two distinct steps shows that these non-native interactions only occur in the final stage of the refolding process in which the two domains match to form the native conformation.     

Detection of a hidden folding intermediate of the third domain of PDZ

The folding pathway of the third domain of PDZ from the synaptic protein PSD-95 was characterized using kinetic and equilibrium methods by monitoring the fluorescence signal from a Trp residue that is incorporated at a near-surface position. Kinetic folding of this domain showed multiple exponential phases, whereas unfolding showed a single exponential phase. The slow kinetic phases were attributed to isomerization of proline residues, since there are five proline residues in this domain. We found that the logarithms of the rate constants for the fast phase of folding and unfolding are linearly dependent on the concentrations of denaturant. The unfolding free energy derived from these rate constants at zero denaturant was close to the value measured using the equilibrium method, suggesting the absence of detectable sub-millisecond folding intermediates. However, native-state hydrogen exchange experiments detected a partially unfolded intermediate under native conditions. It was further confirmed by a protein engineering study. These data suggest that a hidden intermediate exists after the rate-limiting step in the folding of the third domain of PDZ.     

Three-state protein folding: experimental determination of free-energy profile

When considering protein folding with a transient intermediate, a difficulty arises as to determination of the rates of separate transitions. Here we overcome this problem, using the kinetic studies of the unfolding/refolding reactions of the three-state protein apomyoglobin as a model. Amplitudes of the protein refolding kinetic burst phase corresponding to the transition from the unfolded (U) to intermediate (I) state, that occurs prior to the native state (N) formation, allow us to estimate relative populations of the rapidly converting states at various final urea concentrations. On the basis of these proportions, a complicated experimental chevron plot has been deconvolved into the urea-dependent rates of the I<-->N and U<-->N transitions to give the dependence of free energies of the main transition state and of all three (N, I, and U) stable states on urea concentration.     

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.     

An NMR view of the folding process of a CheY mutant at the residue level

The folding of CheY mutant F14N/V83T was studied at 75 residues by NMR. Fluorescence, NMR, and sedimentation equilibrium studies at different urea and protein concentrations reveal that the urea-induced unfolding of this CheY mutant includes an on-pathway molten globule-like intermediate that can associate off-pathway. The populations of native and denatured forms have been quantified from a series of 15N-1H HSQC spectra recorded under increasing concentrations of urea. A thermodynamic analysis of these data provides a detailed picture of the mutant's unfolding at the residue level: (1) the transition from the native state to the molten globule-like intermediate is highly cooperative, and (2) the unfolding of this state is sequential and yields another intermediate showing a collapsed N-terminal domain and an unfolded C-terminal tail. This state presents a striking similarity to the kinetic transition state of the CheY folding pathway.     

Test for cooperativity in the early kinetic intermediate in lysozyme folding

During the folding of many proteins, collapsed globular states are formed prior to the native structure. The role of these states for the folding process has been widely discussed. Comparison with properties of synthetic homo and heteropolymers had suggested that the initial collapse represented a shift of the ensemble of unfolded conformations to more compact states without major energy barriers. We investigated the folding/unfolding transition of a collapsed state, which transiently populates early in lysozyme folding. This state forms within the dead-time of stopped-flow mixing and it has been shown to be significantly more compact and globular than the denaturant-induced unfolded state. We used the GdmCl-dependence of the dead-time signal change to characterize the unfolding transition of the burst phase intermediate. Fluorescence and far-UV CD give identical unfolding curves, arguing for a cooperative two-state folding/unfolding transition between unfolded and collapsed lysozyme. These results show that collapse leads to a distinct state in the folding process, which is separated from the ensemble of unfolded molecules by a significant energy barrier. NMR, fluorescence and small angle X-ray scattering data further show that some local interactions in unfolded lysozyme exist at denaturant concentrations above the coil-collapse transition. These interactions might play a crucial role in the kinetic partitioning between fast and slow folding pathways.     

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

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

Kinetics of folding and unfolding of goat alpha-lactalbumin

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

Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA.

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.     

Exploring subdomain cooperativity in T4 lysozyme II: uncovering the C-terminal subdomain as a hidden intermediate in the kinetic folding pathway

Intermediates along a protein's folding pathway can play an important role in its biology. Previous kinetics studies have revealed an early folding intermediate for T4 lysozyme, a small, well-characterized protein composed of an N-terminal and a C-terminal subdomain. Pulse-labeling hydrogen exchange studies suggest that residues from both subdomains contribute to the structure of this intermediate. On the other hand, equilibrium native state hydrogen experiments have revealed a high-energy, partially unfolded form of the protein that has an unstructured N-terminal subdomain and a structured C-terminal subdomain. To resolve this discrepancy between kinetics and equilibrium data, we performed detailed kinetics analyses of the folding and unfolding pathways of T4 lysozyme, as well as several point mutants and large-scale variants. The data support the argument for the presence of two distinct intermediates, one present on each side of the rate-limiting transition state barrier. The effects of circular permutation and site-specific mutations in the wild-type and circular permutant background, as well as a fragment containing just the C-terminal subdomain, support a model for the unfolding intermediate with an unfolded N-terminal and a folded C-terminal subdomain. Our results suggest that the partially unfolded form identified by native state hydrogen exchange resides on the folded side of the rate-limiting transition state and is, therefore, under most conditions, a "hidden" intermediate.     

Analysis of protein folding by fast protein liquid chromatography. Modular domain folding of gamma-II-crystallin from calf eye-lens

Fast protein liquid chromatography was effectively applied to analyse the folding mechanism of gamma-II-crystallin from calf eye-lens. The protein undergoes a bimodal folding/unfolding transition, according to a three-state model: N in equilibrium I in equilibrium D where N, I, and D stand for the native, intermediate and denatured states (R. Rudolph, R. Siebendritt, G. Nesslauer, A.K. Sharma & R. Jaenicke (1990) Proc. Natl. Acad. Sci. USA 87, 4625-4629). Using Superose 12 HR 10/30, the intermediate with the N-terminal domain intact, and the C-terminal domain unfolded, could be separated from the native protein. The N----I transition is sufficiently slow to allow kinetic measurements, following the variation of the respective peak-heights during denaturation/renaturation. The corresponding relaxation times are in agreement with kinetic data based on the change in fluorescence emission accompanying the N in equilibrium I transition.     

Elucidating quantitative stability/flexibility relationships within thioredoxin and its fragments using a distance constraint model

Numerous quantitative stability/flexibility relationships, within Escherichia coli thioredoxin (Trx) and its fragments are determined using a minimal distance constraint model (DCM). A one-dimensional free energy landscape as a function of global flexibility reveals Trx to fold in a low-barrier two-state process, with a voluminous transition state. Near the folding transition temperature, the native free energy basin is markedly skewed to allow partial unfolded forms. Under native conditions the skewed shape is lost, and the protein forms a compact structure with some flexibility. Predictions on ten Trx fragments are generally consistent with experimental observations that they are disordered, and that complementary fragments reconstitute. A hierarchical unfolding pathway is uncovered using an exhaustive computational procedure of breaking interfacial cross-linking hydrogen bonds that span over a series of fragment dissociations. The unfolding pathway leads to a stable core structure (residues 22-90), predicted to act as a kinetic trap. Direct connection between degree of rigidity within molecular structure and non-additivity of free energy is demonstrated using a thermodynamic cycle involving fragments and their hierarchical unfolding pathway. Additionally, the model provides insight about molecular cooperativity within Trx in its native state, and about intermediate states populating the folding/unfolding pathways. Native state cooperativity correlation plots highlight several flexibly correlated regions, giving insight into the catalytic mechanism that facilitates access to the active site disulfide bond. Residual native cooperativity correlations are present in the core substructure, suggesting that Trx can function when it is partly unfolded. This natively disordered kinetic trap, interpreted as a molten globule, has a wide temperature range of metastability, and it is identified as the "slow intermediate state" observed in kinetic experiments. These computational results are found to be in overall agreement with a large array of experimental data.     

A folding model of α-lactalbumin deduced from the three-state denaturation mechanism

It has been shown that α-lactalbumin undergoes a three-state denaturation, involving a helical intermediate state, on treatment with guanidine hydrochloride. The unfolding of the protein and the characteristics of the intermediate state are examined by means of circular dichroism, difference spectra and pH-jump measurements to investigate the temperature dependence and kinetic properties of the unfolding and refolding, the pH dependence of the transition between the intermediate and the fully unfolded states, and the effect of disulphide bond reduction on the stabilization of the intermediate.The results show that the long-range specific interactions such as specific electrostatic interactions and disulphide linkages are not important for stabilizing the intermediate, and that the transition between the intermediate and the fully unfolded states is extremely rapid (a relaxation time of less than one millisecond) and may correspond to the helix-coil transition of a polypeptide backbone. On the other hand, the activation parameters of the transition between the native and the intermediate states have suggested that the final stabilization by charge-pair interactions is preceded by hydrophobic interactions in the process of going from the intermediate to the native state.The mechanism of folding of the protein is discussed, and the folding process from the fully unfolded to the native state is apparently divided into at least three main steps: (1) the formation of incipient helical structures dictated by local interactions; (2) the packing of the helical segments accompanied with hydrophobic interactions; (3) the final stabilization by the electrostatic interactions. The relevance to the current theoretical results on protein folding is also discussed.