How do chemical denaturants affect the mechanical folding and unfolding of proteins?

We present the first single-molecule atomic force microscopy study on the effect of chemical denaturants on the mechanical folding/unfolding kinetics of a small protein GB1 (the B1 immunoglobulin-binding domain of protein G from Streptococcus). Upon increasing the concentration of the chemical denaturant guanidinium chloride (GdmCl), we observed a systematic decrease in the mechanical stability of GB1, indicating the softening effect of the chemical denaturant on the mechanical stability of proteins. This mechanical softening effect originates from the reduced free-energy barrier between the folded state and the unfolding transition state, which decreases linearly as a function of the denaturant concentration. Chemical denaturants, however, do not alter the mechanical unfolding pathway or shift the position of the transition state for mechanical unfolding. We also found that the folding rate constant of GB1 is slowed down by GdmCl in mechanical folding experiments. By combining the mechanical folding/unfolding kinetics of GB1 in GdmCl solution, we developed the “mechanical chevron plot” as a general tool to understand how chemical denaturants influence the mechanical folding/unfolding kinetics and free-energy diagram in a quantitative fashion. This study demonstrates great potential in combining chemical denaturation with single-molecule atomic force microscopy techniques to reveal invaluable information on the energy landscape underlying protein folding/unfolding reactions.     

Energy barriers, cooperativity, and hidden intermediates in the folding of small proteins

Current theoretical views of the folding process of small proteins (< approximately 100 amino acids) postulate that the landscape of potential mean force (PMF) for the formation of the native state has a funnel shape and that the free energy barrier to folding arises from the chain configurational entropy only. However, recent theoretical studies on the formation of hydrophobic clusters with explicit water suggest that a barrier should exist on the PMF of folding, consistent with the fact that protein folding generally involves a large positive activation enthalpy at room temperature. In addition, high-resolution structural studies of the hidden partially unfolded intermediates have revealed the existence of non-native interactions, suggesting that the correction of the non-native interactions during folding should also lead to barriers on PMF. To explore the effect of a PMF barrier on the folding behavior of proteins, we modified Zwanzig's model for protein folding with an uphill landscape of PMF for the formation of transition states. We found that the modified model for short peptide segments can satisfy the thermodynamic and kinetic criteria for an apparently two-state folding. Since the Levinthal paradox can be solved by a stepwise folding of short peptide segments, a landscape of PMF with a locally uphill search for the transition state and cooperative stabilization of folding intermediates/native state is able to explain the available experimental results for small proteins. We speculate that the existence of cooperative hidden folding intermediates in small proteins could be the consequence of the highly specific structures of the native state, which are selected by evolution to perform specific functions and fold in a biologically meaningful time scale.     

Protein interactions with urea and guanidinium chloride. A calorimetric study.

The interaction of urea and guanidinium chloride with proteins has been studied calorimetrically by titrating protein solutions with denaturants at various fixed temperatures, and by scanning them with temperature at various fixed concentrations of denaturants. It has been shown that the observed heat effects can be described in terms of a simple binding model with independent and similar binding sites. Using the calorimetric data, the number of apparent binding sites for urea and guanidinium chloride have been estimated for three proteins in their unfolded and native states (ribonuclease A, hen egg white lysozyme and cytochrome c). The intrinsic and total thermodynamic characteristics of their binding (the binding constant, the Gibbs energy, enthalpy, entropy and heat capacity effect of binding) have also been determined. It is found that the binding of urea and guanidinium chloride by protein is accompanied by a significant decrease of enthalpy and entropy. At all concentrations of denaturants the enthalpy term slightly dominates the entropy term in the Gibbs energy function. Correlation analysis of the number of binding sites and structural characteristics of these proteins suggests that the binding sites for urea and guanidinium chloride are likely to be formed by several hydrogen bonding groups. This type of binding of the denaturant molecules should lead to a significant restriction of conformational freedom within the polypeptide chain. This raises a doubt as to whether a polypeptide chain in concentrated solutions of denaturants can be considered as a standard of a random coil conformation.     

Water contributes actively to the rapid crossing of a protein unfolding barrier

The cold-shock protein CspB folds rapidly in a N <= => U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive (Delta V(++)(NU)=16(+/-4) ml/mol) and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well (Delta V(++)(NU)=53(+/-6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, Delta V(++)(UN) decreases significantly, suggesting that compact unfolded forms become populated under these conditions.     

Kinetic analysis of folding and unfolding the 56 amino acid IgG-binding domain of streptococcal protein G.

The 56 amino acid B domain of protein G (GB) is a stable globular folding unit with no disulfide cross-links. The physical properties of GB offer extraordinary flexibility for evaluating the energetics of the folding reaction. The protein is monomeric and very soluble in both folded and unfolded forms. The folding reaction has been previously examined by differential scanning calorimetry (Alexander et al., 1992) and found to exhibit two-state unfolding behavior over a wide pH range with an unfolding transition near 90 degrees C (GB1) at neutral pH. Here, the kinetics of folding and unfolding two naturally occurring versions of GB have been measured using stopped-flow mixing methods and analyzed according to transition-state theory. GB contains no prolines, and the kinetics of folding and unfolding can be fit to a single, first-order rate constant over the temperature range of 5-35 degrees C. The major thermodynamic changes going from the unfolded state to the transition state are (1) a large decrease in heat capacity (delta Cp), indicating that the transition state is compact and solvent inaccessible relative to the unfolded state; (2) a large loss of entropy; and (3) a small increase in enthalpy. The most surprising feature of the folding of GB compared to that of previously studied proteins is that its folding approximates a rapid diffusion controlled process with little increase in enthalpy going from the unfolded to the transition state.     

Non-linear effects of temperature and urea on the thermodynamics and kinetics of folding and unfolding of hisactophilin

Extensive measurements and analysis of thermodynamic stability and kinetics of urea-induced unfolding and folding of hisactophilin are reported for 5-50 degrees C, at pH 6.7. Under these conditions hisactophilin has moderate thermodynamic stability, and equilibrium and kinetic data are well fit by a two-state transition between the native and the denatured states. Equilibrium and kinetic m values decrease with increasing temperature, and decrease with increasing denaturant concentration. The betaF values at different temperatures and urea concentrations are quite constant, however, at about 0.7. This suggests that the transition state for hisactophilin unfolding is native-like and changes little with changing solution conditions, consistent with a narrow free energy profile for the transition state. The activation enthalpy and entropy of unfolding are unusually low for hisactophilin, as is also the case for the corresponding equilibrium parameters. Conventional Arrhenius and Eyring plots for both folding and unfolding are markedly non-linear, but these plots become linear for constant DeltaG/T contours. The Gibbs free energy changes for structural changes in hisactophilin have a non-linear denaturant dependence that is comparable to non-linearities observed for many other proteins. These non-linearities can be fit for many proteins using a variation of the Tanford model, incorporating empirical quadratic denaturant dependencies for Gibbs free energies of transfer of amino acid constituents from water to urea, and changes in fractional solvent accessible surface area of protein constituents based on the known protein structures. Noteworthy exceptions that are not well fit include amyloidogenic proteins and large proteins, which may form intermediates. The model is easily implemented and should be widely applicable to analysis of urea-induced structural transitions in proteins.     

Thermodynamics of a diffusional protein folding reaction

The folding reactions of several proteins are well described as diffusional barrier crossing processes, which suggests that they should be analyzed by Kramers' rate theory rather than by transition state theory. For the cold shock protein Bc-Csp from Bacillus caldolyticus, we measured stability and folding kinetics, as well as solvent viscosity as a function of temperature and denaturant concentration. Our analysis indicates that diffusional folding reactions can be treated by transition state theory, provided that the temperature and denaturant dependence of the solvent viscosity is properly accounted for, either at the level of the measured rate constants or of the calculated activation parameters. After viscosity correction the activation barriers for folding become less enthalpic and more entropic. The transition from an enthalpic to an entropic folding barrier with increasing temperature is, however, apparent in the data before and after this correction. It is a consequence of the negative activation heat capacity of refolding, which is independent of solvent viscosity. Bc-Csp and its mesophilic homolog Bs-CspB from Bacillus subtilis differ strongly in stability but show identical enthalpic and entropic barriers to refolding. The increased stability of Bc-Csp originates from additional enthalpic interactions that are established after passage through the activated state. As a consequence, the activation enthalpy of unfolding is increased relative to Bs-CspB.     

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.     

Solvent denaturation and stabilization of globular proteins

Statistical thermodynamic theory has recently been developed to account for the stabilities of globular proteins. Here we extend that work to predict the effects of solvents on protein stability. Folding is assumed to be driven by solvophobic interactions and opposed by conformational entropy. The solvent dependence of the solvophobic interactions is taken from transfer experiments of Nozaki and Tanford on amino acids into aqueous solutions of urea or guanidine hydrochloride (GuHCl). On the basis of the assumption of two pathways involving collapse and formation of a core, the theory predicts that increasing denaturant should lead to a two-state denaturation transition (i.e., there is a stable state along each path separated by a free energy barrier). The denaturation midpoint is predicted to occur at higher concentrations of urea than of GuHCl. At neutral pH, the radius of the solvent-denatured state should be much smaller than for a random-flight chain and increase with either denaturant concentration or number of polar residues in the chain. A question of interest is whether free energies of folding should depend linearly on denaturant, as is often assumed. The free energy is predicted to be linear for urea but to have some small curvature for GuHCl. Predicted slopes and exposed areas of the unfolded states are found to be in generally good agreement with experiments. We also discuss stabilizing solvents and compare thermal with solvent denaturation.     

The nature of the free energy barriers to two-state folding

We introduce a simple procedure to analyze the temperature dependence of the folding and unfolding rates of two-state proteins. We start from the simple transition-state-like rate expression: k = D(eff)exp(-DeltaG(TS)/RT), in which upper and lower bounds for the intra-chain effective diffusion coefficient (D(eff)) are obtained empirically using the timescales of elementary processes in protein folding. From the changes in DeltaG(TS) as a function of temperature, we calculate enthalpies and heat capacities of activation, together with the more elusive entropies of activation. We then estimate the conformational entropy of the transition state by extrapolation to the temperature at which the solvation entropy vanishes by cancellation between polar and apolar terms. This approach is based on the convergence temperatures for the entropy of solvating apolar (approximately 385 K) and polar groups (approximately 335 K), the assumption that the structural properties of the transition state are somewhere in between the unfolded and folded states, and the established relationship between observed heat capacity and solvent accessibility.1 To circumvent the lack of structural information about transition states, we use the empirically determined heat capacities of activation as constraints to identify the extreme values of the transition state conformational entropy that are consistent with experiment. The application of this simple approach to six two-state folding proteins for which there is temperature-dependent data available in the literature provides important clues about protein folding. For these six proteins, we obtain an average equilibrium cost in conformational entropy of -4.3 cal x mol(-1)K(-1)per residue, which is in close agreement to previous empirical and computational estimates of the same quantity. Furthermore, we find that all these proteins have a conformationally diverse transition state, with more than half of the conformational entropy of the unfolded state. In agreement with predictions from theory and computer simulations, the transition state signals the change from a regime dominated by loss in conformational entropy to one driven by the gain in stabilization free energy (i.e., including protein interactions and solvation effects). Moreover, the height of the barrier is determined by how much stabilization free energy is realized at that point, which is related to the relative contribution of local versus non-local interactions. A remarkable observation is that the fraction of conformational entropy per residue that is present in the transition state is very similar for the six proteins in this study. Based on this commonality, we propose that the observed change in thermodynamic regime is connected to a change in the pattern of structure formation: from one driven by formation of pairwise interactions to one dominated by coupling of the networks of interactions involved in forming the protein core. In this framework, the barrier to two-state folding is crossed when the folding protein reaches a "critical native density" that allows expulsion of remaining interstitial water and consolidation of the core. The principle of critical native density should be general for all two-state proteins, but can accommodate different folding mechanisms depending on the particularities of the structure and sequence.     

Shape of the free energy barriers for protein folding probed by multiple perturbation analysis

The characterization of the free energy barriers has been a major goal in studies on the mechanism of protein folding. Testing the effect of mutations or denaturants on protein folding reactions revealed that transition state movement is rare, suggesting that folding barriers are robust and narrow maxima on the free energy landscape. Here we demonstrate that the application of multiple perturbations allows the observation of small transition state movements that escape detection in single perturbation experiments. We used tendamistat as a model protein to test the broadness of the free energy barriers. Tendamistat folds over two consecutive transition states and through a high-energy intermediate. Measuring the combined effect of temperature and denaturant on the position of the transition state in the wild-type protein and in several mutants revealed that the early transition state shows significant transition state movement. Its accessible surface area state becomes more native-like with destabilization of the native state by temperature. To the same extent, the entropy of the early transition state becomes more native-like with increasing denaturant concentration, in accordance with Hammond behavior. The position of the late transition state, in contrast, is much less sensitive to the applied perturbations. These results suggest that the barriers in protein folding become increasingly narrow as the folding polypeptide chain approaches the native state.     

Free energy landscape and folding mechanism of a beta-hairpin in explicit water: a replica exchange molecular dynamics study

The free energy landscape and the folding mechanism of the C-terminal beta-hairpin of protein G is studied by extensive replica exchange molecular dynamics simulations (40 replicas and 340 ns total simulation time), using the GROMOS96 force field and the SPC explicit water solvent. The study reveals that the system preferentially adopts a beta-hairpin structure at biologically important temperatures, and that the helix content is low at all temperatures studied. Representing the free energy landscape as a function of several types of reaction coordinates, four local minima corresponding to the folded, partially folded, molten globule, and unfolded states are identified. The findings suggest that the folding of the beta-hairpin occurs as the sequence: collapse of hydrophobic core --> formation of H-bond --> formation of the turn. Identifying the folded and molten globule states as the main conformations, the free energy landscape of the beta-hairpin is consistent with a two-state behavior with a broad transition state. The temperature dependence of the folding-unfolding transition is investigated in some detail. The enthalpy and entropy jumps at the folding transition temperature are found to be about three times lower than the experimental estimates, indicating that the folding-unfolding transition in silico is less cooperative than its in vitro counterpart.     

Effects of urea and guanidine · HCl on the folding and unfolding of pancreatic trypsin inhibitor

Concentrated solutions of urea and of guanidine · HCl produced a random spectrum of single-disulphide forms of the polypeptide chain of the pancreatic trypsin inhibitor. Guanidine · HCl also unfolded completely, with accompanying interchange of disulphide bonds, the two-disulphide form of this protein in the native-like conformation; urea produced an equilibrium mixture in which one-quarter of the molecules had the native-like conformation and disulphide bonds. The unfolded forms of the protein in the denaturants were very flexible polypeptide chains. The observations suggest that urea and guanidine · HCl are denaturants because they produce essentially equally favourable solvation of all portions of a polypeptide.The energetics of the conformational transitions involved in folding and unfolding of the inhibitor were determined in urea and compared with those observed in its absence. The denaturant lowers the stability of the native, folded inhibitor relative to that of the reduced, unfolded state by 6.5 kilocalories per mole; the greatest part of this apparent free-energy difference was expressed at the two-disulphide stage of folding. The results are consistent with other indications that most of the favourable interactions stabilizing the native conformation of this protein are not encountered until the final stage of folding, when all may occur simultaneously.The unfolded one- and two-disulphide species produced in guanidine · HCl were trapped, and their rearrangement to the normal intermediates followed after removal of the denaturant. The random single-disulphide species, with one exception, reverted very rapidly to the non-random spectrum of intermediates normally observed during folding; this confirms that these species are normally rapidly interconverted and that normal refolding of the reduced protein is not dependent kinetically upon residual stable conformation in the reduced protein. The unfolded two-disulphide species refolded to the native-like conformation more slowly, but appeared to pass through the same intermediates normally observed during refolding from the fully reduced state.     

Relationship between the native-state hydrogen exchange and folding pathways of a four-helix bundle protein

The hydrogen exchange behavior of a four-helix bundle protein in low concentrations of denaturant reveals some partially unfolded forms that are significantly more stable than the fully unfolded state. Kinetic folding of the protein, however, is apparently two-state in the absence of the accumulation of early folding intermediates. The partially unfolded forms are either as folded as or more folded than the rate-limiting transition state and appear to represent the major intermediates in a folding and unfolding reaction. These results are consistent with the suggestion that partially unfolded intermediates may form after the rate-limiting step for small proteins with apparent two-state folding kinetics.     

Molecular basis for the effect of urea and guanidinium chloride on the dynamics of unfolded polypeptide chains

Chemical denaturants are frequently used to unfold proteins and to characterize mechanisms and transition states of protein folding reactions. The molecular basis of the effect of urea and guanidinium chloride (GdmCl) on polypeptide chains is still not well understood. Models for denaturant--protein interaction include both direct binding and indirect changes in solvent properties. Here we report studies on the effect of urea and GdmCl on the rate constants (k(c)) of end-to-end diffusion in unstructured poly(glycine-serine) chains of different length. Urea and GdmCl both lead to a linear decrease of lnk(c) with denaturant concentration, as observed for the rate constants for protein folding. This suggests that the effect of denaturants on chain dynamics significantly contributes to the denaturant-dependence of folding rate constants for small proteins. We show that this linear dependency is the result of two additive non-linear effects, namely increased solvent viscosity and denaturant binding. The contribution from denaturant binding can be quantitatively described by Schellman's weak binding model with binding constants (K) of 0.62(+/-0.01)M(-1) for GdmCl and 0.26(+/-0.01)M(-1) for urea. In our model peptides the number of binding sites and the effect of a bound denaturant molecule on chain dynamics is identical for urea and GdmCl. The results further identify the polypeptide backbone as the major denaturant binding site and give an upper limit of a few nanoseconds for residence times of denaturant molecules on the polypeptide chain.     

How cooperative are protein folding and unfolding transitions?

A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two‐state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non‐cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier‐less “downhill” folding, as well as for continuous “uphill” unfolding transitions, indicate that gradual non‐cooperative processes may be ubiquitous features on the free energy landscape of protein folding.     

Molecular basis of cooperativity in protein folding IV. Core: A general cooperative folding model

The cooperative nature of the protein folding process is independent of the characteristic fold and the specific secondary structure attributes of a globular protein. A general folding/unfolding model should, therefore, be based upon structural features that transcend the peculiarities of α-helices, β-sheets, and other structural motifs found in proteins. The studies presented in this paper suggest that a single structural characteristic common to all globular proteins is essential for cooperative folding. The formation of a partly folded state from the native state results in the exposure to solvent of two distinct regions: (1) the portions of the protein that are unfolded; and (2) the “complementary surfaces,” located in the regions of the protein that remain folded. The cooperative character of the folding/unfolding transition is determined largely by the energetics of exposing complementary surface regions to the solvent. By definition, complementary regions are present only in partly folded states; they are absent from the native and unfolded states. An unfavorable free energy lowers the probability of partly folded states and increases the cooperativity of the transition. In this paper we present a mathematical formulation of this behavior and develop a general cooperative folding/unfolding model, termed the “complementary region” (CORE) model. This model successfully reproduces the main properties of folding/unfolding transitions without limiting the number of partly folded states accessible to the protein, thereby permitting a systematic examination of the structural and solvent conditions under which intermediates become populated. It is shown that the CORE model predicts two-state folding/unfolding behavior, even though the two-state character is not assumed in the model. © 1993 Wiley-Liss, Inc.     

Unfolding rates of barstar determined in native and low denaturant conditions indicate the presence of intermediates

The folding and unfolding rates of the small protein, barstar, have been monitored using stopped-flow measurements of intrinsic tryptophan fluorescence at 25 degrees C, pH 8.5, and have been compared over a wide range of urea and guanidine hydrochloride (GdnHCl) concentrations. When the logarithms of the rates of folding from urea and from GdnHCl unfolded forms are extrapolated linearly with denaturant concentration, the same rate is obtained for folding in zero denaturant. Similar linear extrapolations of rates of unfolding in urea and GdnHCl yield, however, different unfolding rates in zero denaturant, indicating that such linear extrapolations are not valid. It has been difficult, for any protein, to determine unfolding rates under nativelike conditions in direct kinetic experiments. Using a novel strategy of coupling the reactivity of a buried cysteine residue with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to the unfolding reaction of barstar, the global unfolding and refolding rates have now been determined in low denaturant concentrations. The logarithms of unfolding rates obtained at low urea and GdnHCl concentrations show a markedly nonlinear dependence on denaturant concentration and converge to the same unfolding rate in the absence of denaturant. It is shown that the native protein can sample the fully unfolded conformation even in the absence of denaturant. The observed nonlinear dependences of the logarithms of the refolding and unfolding rates observed for both denaturants are shown to be due to the presence of (un)folding intermediates and not due to movements in the position of the transition state with a change in denaturant concentration.     

Dimensions,energetics, and denaturant effects of the protein unstructured state

Determining the energetics of the unfolded state of a protein is essential for understanding the folding mechanics of ordered proteins and the structure–function relation of intrinsically disordered proteins. Here, we adopt a coil‐globule transition theory to develop a general scheme to extract interaction and free energy information from single‐molecule fluorescence resonance energy transfer spectroscopy. By combining protein stability data, we have determined the free energy difference between the native state and the maximally collapsed denatured state in a number of systems, providing insight on the specific/nonspecific interactions in protein folding. Both the transfer and binding models of the denaturant effects are demonstrated to account for the revealed linear dependence of inter‐residue interactions on the denaturant concentration, and are thus compatible under the coil‐globule transition theory to further determine the dimension and free energy of the conformational ensemble of the unfolded state. The scaling behaviors and the effective θ‐state are also discussed.     

The folding pathway of barnase: the rate-limiting transition state and a hidden intermediate under native conditions

The nature of the rate-limiting transition state at zero denaturant (TS(1)) and whether there are hidden intermediates are the two major unsolved problems in defining the folding pathway of barnase. In earlier studies, it was shown that TS(1) has small phi values throughout the structure of the protein, suggesting that the transition state has either a defined partially folded secondary structure with all side chains significantly exposed or numerous different partially unfolded structures with similar stability. To distinguish the two possibilities, we studied the effect of Gly mutations on the folding rate of barnase to investigate the secondary structure formation in the transition state. Two mutations in the same region of a beta-strand decreased the folding rate by 20- and 50-fold, respectively, suggesting that the secondary structures in this region are dominantly formed in the rate-limiting transition state. We also performed native-state hydrogen exchange experiments on barnase at pD 5.0 and 25 degrees C and identified a partially unfolded state. The structure of the intermediate was investigated using protein engineering and NMR. The results suggest that the intermediate has an omega loop unfolded. This intermediate is more folded than the rate-limiting transition state previously characterized at high denaturant concentrations (TS(2)). Therefore, it exists after TS(2) in folding. Consistent with this conclusion, the intermediate folds with the same rate and denaturant dependence as the wild-type protein, but unfolds faster with less dependence on the denaturant concentration. These and other results in the literature suggest that barnase folds through partially unfolded intermediates that exist after the rate-limiting step. Such folding behavior is similar to those of cytochrome c and Rd-apocyt b(562). Together, we suggest that other small apparently two-state proteins may also fold through hidden intermediates.