Molecular basis of co-operativity in protein folding.

The folding/unfolding transition of proteins is a highly co-operative process characterized by the presence of very few or no thermodynamically stable partially folded intermediate states. The purpose of this paper is to present a thermodynamic formalism aimed at describing quantitatively the co-operative folding behavior of proteins. In order to account for this behavior, a hierarchical algorithm aimed at evaluating the folding/unfolding partition function has been developed. This formalism defines the partition function in terms of multiple levels of interacting co-operative folding units. A co-operative folding unit is defined as a protein structural element that exhibits two-state folding/unfolding behavior. At the most fundamental level are those structural elements that behave co-operatively as a result of purely local interactions. Higher-order co-operative folding units are formed through interactions between different structural elements. The hierarchical formalism utilizes the crystallographic structure of the protein as a template to generate partially folded conformations defined in terms of co-operative folding units. The Gibbs free energy of those states and their corresponding statistical weights are then computed using experimental energetic parameters determined calorimetrically. This formalism has been applied to the case of myoglobin. It is shown that the hierarchical partition function correctly predicts the presence, energetics and co-operativity of the heat and cold denaturation transitions. The major contribution to the co-operative folding behavior arises from the solvent exposure of non-polar residues located in regions complementary to those that have undergone unfolding. This entropically uncompensated and energetically unfavorable solvent exposure characterizes all partially folded states but not the unfolded state, thus minimizing the population of partially folded intermediates throughout the folding/unfolding transition.     

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.     

An insight into domain structures and thermal stability of gamma-crystallins.

The thermal behavior of gamma II, gamma IIIA, gamma IIIB, and gamma IVA crystallin, from calorimetric and spectral studies, has been analyzed in terms of selective unfolding of domains, interdomain interactions, conformational stability, and the existence of intermediates in the order-disorder transition equilibrium. The major endothermic transition (Tm) observed calorimetrically for all four fractions occurs between 67 and 78 degrees C, with enthalpy change (delta H) from 80 to 150 kcal/mol, values that agree reasonably well with those from spectroscopic measurements. gamma II and gamma IIIB show a second thermal event at T less than Tm whereas gamma IIIA and gamma IVA showed no additional transition. Urea-induced equilibrium unfolding of gamma II at acidic pH, unlike gamma IVA, is biphasic as monitored by CD and fluorescence, indicating the existence of an intermediate. The absence of a cooperative transition in gamma IVA in acidic urea and the appearance of a single endotherm in differential scanning calorimetry at low pH have been attributed to a structured intermediate that melts at low temperature. The difference in the folding/unfolding of gamma II and gamma IVA has been explained by subtle differences in the packing arrangement of their two domains and interactions between them. Thermal aggregation of gamma-crystallins could be prevented either by preincubation with ionic detergents or at low pH or in the presence of chemical denaturant, indicating that the protein surface charge and solvent polarity influence their stability. An increase in the 8-anilino-1-naphthalenesulfonate-bound fluorescence during heat denaturation also suggests that the thermal aggregation is governed by hydrophobic interactions.     

Criteria for downhill protein folding: calorimetry, chevron plot, kinetic relaxation, and single-molecule radius of gyration in chain models with subdued degrees of cooperativity

Recent investigations of possible downhill folding of small proteins such as BBL have focused on the thermodynamics of non-two-state, "barrierless" folding/denaturation transitions. Downhill folding is noncooperative and thermodynamically "one-state," a phenomenon underpinned by a unimodal conformational distribution over chain properties such as enthalpy, hydrophobic exposure, and conformational dimension. In contrast, corresponding distributions for cooperative two-state folding are bimodal with well-separated population peaks. Using simplified atomic modeling of a three-helix bundle-in a scheme that accounts for hydrophobic interactions and hydrogen bonding-and coarse-grained C(alpha) models of four real proteins with various degrees of cooperativity, we evaluate the effectiveness of several observables at defining the underlying distribution. Bimodal distributions generally lead to sharper transitions, with a higher heat capacity peak at the transition midpoint, compared with unimodal distributions. However, the observation of a sigmoidal transition is not a reliable criterion for two-state behavior, and the heat capacity baselines, used to determine the van't Hoff and calorimetric enthalpies of the transition, can introduce ambiguity. Interestingly we find that, if the distribution of the single-molecule radius of gyration were available, it would permit discrimination between unimodal and bimodal underlying distributions. We investigate kinetic implications of thermodynamic noncooperativity using Langevin dynamics. Despite substantial chevron rollovers, the relaxation of the models considered is essentially single-exponential over an extended range of native stabilities. Consistent with experiments, significant deviations from single-exponential behavior occur only under strongly folding conditions.     

Thermodynamics of barnase unfolding.

The thermodynamics of barnase denaturation has been studied calorimetrically over a broad range of temperature and pH. It is shown that in acidic solutions the heat denaturation of barnase is well approximated by a 2-state transition. The heat denaturation of barnase proceeds with a significant increase of heat capacity, which determines the temperature dependencies of the enthalpy and entropy of its denaturation. The partial specific heat capacity of denatured barnase is very close to that expected for the completely unfolded protein. The specific denaturation enthalpy value extrapolated to 130 degrees C is also close to the value expected for the full unfolding. Therefore, the calorimetrically determined thermodynamic characteristics of barnase denaturation can be considered as characteristics of its complete unfolding and can be correlated with structural features--the number of hydrogen bonds, extent of van der Waals contacts, and the surface areas of polar and nonpolar groups. Using this information and thermodynamic information on transfer of protein groups into water, the contribution of various factors to the stabilization of the native structure of barnase has been estimated. The main contributors to the stabilization of the native state of barnase appear to be intramolecular hydrogen bonds. The contributions of van der Waals interactions between nonpolar groups and those of hydration effects of these groups are not as large if considered separately, but the combination of these 2 factors, known as hydrophobic interactions, is of the same order of magnitude as the contribution of hydrogen bonding.     

Burst-phase expansion of native protein prior to global unfolding in SDS

Although numerous studies have been directed at understanding early folding events through the characterization of folding intermediates, there are few reports on the very late folding events, i.e. on the events taking place on the native side of the folding barrier and on alternative conformations of the folded state. To shed further light on these issues, we have characterized by protein engineering the structure of an expanded but native-like intermediate that accumulates transiently in the unfolding reaction of the small protein S6 in the presence of SDS. The results show that the SDS micelles attack the native protein in the dead-time of the denaturation experiment, causing an expansion of the hydrophobic core prior to the major unfolding transition. We distinguish two forms of the unfolding intermediate that are correlated with the micellar structure. With spherical micelles, the expansion is seen mainly as a weakening of the interactions which anchor the two alpha-helices to the core of the S6 structure. With cylindrical micelles, prevalent at higher SDS concentrations, the expansion is more global and produces a species which closely resembles the transition-state structure for unfolding in GdmCl. Despite the highly weakened core, the micelle-associated intermediate displays cooperative unfolding, indicating a significant structural plasticity of the species on the native side of the folding barrier in the presence of SDS.     

Heat and cold denaturation of phosphoglycerate kinase (interaction of domains)

It has been shown that the denaturation of phosphoglycerate kinase (PGK) can be observed not only when the solution is heated above 30 degrees C, but also when it is cooled below this temperature. The disruption of the native PGK structure upon cooling and the subsequent formation of this structure upon heating both proceed in two distinct stages which correspond to the independent disruption or reformation of each of its domains. In contrast, the heat denaturation of PGK proceeds in one stage, showing that the two domains of the molecule are associated into a single complex which figures in the denaturation process as a cooperative unit. It follows that, at elevated temperature, there is a positive interaction between the domains, which disappears at lower temperatures. This might be due to hydrophobic interactions, which are known to be temperature dependent. The temperature decrease leads to a decrease in inter- and intradomain interactions, which results in an increase of the independence of the domains and a decrease in their stability.     

Thermodynamic study of phosphoglycerate kinase from Thermotoga maritima and its isolated domains: reversible thermal unfolding monitored by differential scanning calorimetry and circular dichroism spectroscopy

The folding of phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima and its isolated N- and C-terminal domains (N1/2 and C1/2) was characterized by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. At pH 3.0-4.0, reversible thermal denaturation of TmPGK occurred below 90 degrees C. The corresponding peaks in the partial molar heat capacity function were fitted by a four-state model, describing three well-defined unfolding transitions. Using CD spectroscopy, these are ascribed to the disruption of the domain interactions and subsequent sequential unfolding of the two domains. The isolated N-terminal domain unfolds reversibly between pH 3.0 and pH 4.0 to >90% and at pH 7.0 to about 70%. In contrast, the isolated engineered C-terminal domain only shows reversible thermal denaturation between pH 3.0 and pH 3.5. Neither N1/2 nor C1/2 obeys the simple two-state mechanism of unfolding. Instead, both unfold via a partially structured intermediate. In the case of N1/2, the intermediate exhibits native secondary structure and perturbed tertiary structure, whereas for C1/2 the intermediate could not be defined with certainty.     

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.     

Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A

This research was undertaken to distinguish between local and global unfolding in the reversible thermal denaturation of bovine pancreatic ribonclease A (RNase A). Local unfolding was monitored by steady-state and time-resolved fluorescence of nine mutants in each of which a single tryptophan was substituted for a wild-type residue. Global unfolding was monitored by far-UV circular dichroism and UV absorbance. All the mutants (except F8W and D38W) exhibited high specific enzymatic activity, and their far-UV CD spectra were very close to that of wild-type RNase A, indicating that the tryptophan substitutions did not affect the structure of any of the mutants (excluding K1W and Y92W) under folding conditions at 20 degrees C. Like wild-type RNase A, the various mutants exhibited reversible cooperative thermal unfolding transitions at pH 5, with transition temperatures 2.5-11 degrees C lower than that of the wild-type transition, as detected by far-UV CD or UV absorbance. Even at 80 degrees C, well above the cooperative transition of all the RNase A mutants, a considerable amount of secondary and tertiary structure was maintained. These studies suggest the following two-stage mechanism for the thermal unfolding transition of RNase A as the temperature is increased. First, at temperatures lower than those of the main cooperative transition, long-range interactions within the major hydrophobic core are weakened, e.g., those involving residues Phe-8 (in the N-terminal helix) and Lys-104 and Tyr-115 (in the C-terminal beta-hairpin motif). The structure of the chain-reversal loop (residues 91-95) relaxes in the same temperature range. Second, the subsequent higher-temperature cooperative unfolding transition is associated with a loss of secondary structure and additional changes in the tertiary contacts of the major hydrophobic core, e.g., those involving residues Tyr-73, Tyr-76, and Asp-38 on the other side of the molecule. The hydrophobic interactions of the C-terminal loop of the protein are enhanced by high temperature, and perhaps are responsible for the preservation of the local structural environment of Trp-124 at temperatures slightly above the major cooperative transition. The results shed new light on the thermal unfolding transitions, generally supporting the thermal unfolding hypothesis of Burgess and Scheraga, as modified by Matheson and Scheraga.     

The rate of equine liver alcohol dehydrogenase denaturation by urea. Dependence on temperature and denaturant concentration

The kinetics of the irreversible urea denaturation of equine liver alcohol dehydrogenase have been studied as a function of temperature and urea concentration. The unfolding of the macromolecule, monitored by means of the phosphorescence properties of a deeply buried tryptophan residue, was found to be strictly a two-state process over the entire temperature range. It is characterized by a steep dependence on urea concentration typical of highly cooperative transitions and below room temperature it possesses large negative activation energies. The reaction is comparatively slow, does not seem to be preceded by a fast phase, and the rate-limiting step does not have the characteristics of proline isomerization. When the data are analyzed in terms of binding equilibria the temperature dependence results from an anomalously large change in heat capacity. Although this is a property of strong hydrophobic interactions in model compounds the slow rates of denaturation are best understood with a model of protein stability which emphasizes the cooperative nature of intramolecular interactions such as hydrogen bonding.     

Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures.

We have cloned, expressed, and characterized two naturally occurring variations of the IgG-binding domain of streptococcal protein G. The domain is a stable cooperative folding unit of 56 amino acids, which maintains a unique folded structure without disulfide cross-links or tight ligand binding. We have studied the thermodynamics of the unfolding reaction for the two versions of this domain, designated B1 and B2, which differ by six amino acids. They have denaturation temperatures of 87.5 degrees C and 79.4 degrees C, respectively at pH 5.4, as determined by differential scanning calorimetry. Thermodynamic state functions for the unfolding reaction (delta G, delta H, delta S, and delta Cp) have been determined and reveal several interesting insights into the behavior of very small proteins. First, though the B1 domain has a heat denaturation point close to 90 degrees C, it is not unusually stable at physiologically relevant temperatures (delta G = 25 kJ/mol at 37 degrees C). This behavior occurs because the stability profile (delta G vs temperature) is flat and shallow due to the small delta S and delta Cp for unfolding. Related to this point is the second observation that small changes in the free energy of unfolding of the B-domain due to mutation or change in solvent conditions lead to large shifts in the heat denaturation temperature. Third, the magnitude and relative contributions of hydrophobic vs nonhydrophobic forces (per amino acid residue) to the total free energy of folding of the B-domain are remarkably typical of other globular proteins of much larger size.     

The stability, structural organization, and denaturation of pectate lyase C, a parallel beta-helix protein

Pectate lyase C (pelC) was the first protein in which the parallel beta-helix structure was recognized. The unique features of parallel beta-helix-containing proteins-a relatively simple topology and unusual interactions among side chains-make pelC an interesting protein to study with respect to protein folding. In this paper, we report studies of the unfolding equilibrium of pelC. PelC is unfolded reversibly by gdn-HCl at pH 7 and 5, as monitored by far- and near-UV CD and fluorescence. The coincidence of these spectroscopically detected transitions is consistent with a two-state transition at pH 7, but the three probes are not coincident at pH 5. No evidence was found for a loosely folded intermediate in the transition region at pH 5. At pH 7, the for unfolding is 12.2 kcal/mol, with the midpoint of the transition at 0.99 M gdn-HCl and m = 12.3 kcal/(mol.M). Thus, pelC is unusually stable and has an m value that is much larger than for typical globular proteins. Thermal denaturation of pelC has been studied by differential scanning calorimetry (DSC) and by CD. Although thermal denaturation is not reversible, valid thermodynamic data can be obtained for the unfolding transition. DeltaH(van't Hoff)/DeltaH(cal) is less than 1 for pHs between 5 and 8, with a maximum value of 0.91 at pH 7 decreasing to 0.85 at pH 8 and to 0.68 at pH 5. At all pHs studied, the excess heat capacity can be deconvoluted into two components corresponding to two-state transitions that are nearly coincident at pH 7, but deviate more at higher and lower pH. Thus, pelC appears to consist of two domains that interact strongly and unfold in a cooperative fashion at pH 7, but the cooperativity decreases at higher and lower pH. The crystal structure of pelC shows no obvious domain structure, however.     

Equilibrium and kinetics of the thermal unfolding of alpha-lactalbumin. The relation to its folding mechanism.

The thermal unfolding of alpha-lactalbumin has been studied by equilibrium measurements of aromatic difference spectra, and by kinetic measurements of the Joule heating temperature-jump. The unfolding at neutral pH is a reversible two-state transition. The equilibrium transition curves are analyzed by the nonlinear squares method, which gives correct values of thermodynamic parameters based on the data in a wide range of temperature. The results are discussed in relation to the previous studies on the unfolding by guanidine hydrochloride or by acid. The thermally unfolded state, a partially unfolded species, is shown to be thermodynamically similar to but not identical with the acid state. The folding pathway deduced from the kinetic results is essentially consistent with the folding model of alpha-lactalbumin proposed previously. Large decreases in entropy and in heat capacity during the reversed activation suggest the packing of the folded segments by hydrophobic interactions, while the forward activation shows a marked temperature dependence, probably caused by the disruption of specific long-range interactions.     

Elimination of the C-cap in ubiquitin - structure, dynamics and thermodynamic consequences

Single amino acid substitutions rarely produce substantial changes in protein structure. Here we show that substitution of the C-cap residue in the alpha-helix of ubiquitin with proline (34P variant) leads to dramatic structural changes. The resulting conformational perturbation extends over the last two turns of the alpha-helix and leads to enhanced flexibility for residues 27-37. Thermodynamic analysis of this ubiquitin variant using differential scanning calorimetry reveals that the thermal unfolding transition remains highly cooperative, exhibiting two-state behavior. Similarities with the wild type in the thermodynamic parameters (heat capacity change upon unfolding and m-value) of unfolding monitored by DSC and chemical denaturation suggests that the 34P variant has comparable buried surface area. The hydrophobic core of 34P variant is not packed as well as that of the wild type protein as manifested by a lower enthalpy of unfolding. The increased mobility of the polypeptide chain of this ubiquitin variant allows the transient opening of the hydrophobic core as evidenced by ANS binding. Taken together, these results suggest exceptional robustness of cooperativity in protein structures.     

Thermodynamic studies of the core histones: stability of the octamer subunits is not altered by removal of their terminal domains.

We have investigated the role of the labile terminal domains of the core histones on the stability of the subunits of the protein core of the nucleosome by studying the thermodynamic behavior of the products of limited trypsin digestion of these subunits. The thermal stabilities of the truncated H2A-H2B dimer and the truncated (H3-H4)/(H3-H4)(2) system were studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. The thermal denaturation of the truncated H2A-H2B dimer at pH 6.0 and low ionic strength is centered at 47.3 degrees C. The corresponding enthalpy change is 35 kcal/mol of 11.5 kDa monomer unit, and the heat capacity change upon unfolding is 1.2 kcal/(K mol of 11.5 kDa monomer unit). At pH 4.5 and low ionic strength, the truncated (H3-H4)/(H3-H4)(2) system, like its full-length counterpart, is quantitatively dissociated into two truncated H3-H4 dimers. The thermal denaturation of the truncated H3-H4 dimer is characterized by the presence of a single calorimetric peak centered at 60 degrees C. The enthalpy change is 25 kcal/mol of 10 kDa monomer unit, and the change in heat capacity upon unfolding is 0.5 kcal/(K mol of 10 kDa monomer unit). The thermal stabilities of both types of truncated dimers exhibit salt and pH dependencies similar to those of the full-length proteins. Finally, like their full-length counterparts, both truncated core histone dimers undergo thermal denaturation as highly cooperative units, without the involvement of any significant population of melting intermediates. Therefore, removal of the histone "tails" does not generally affect the thermodynamic behavior of the subunits of the core histone complex, indicating that the more centrally located regions of the histone fold and the extra-fold structured elements are primarily responsible for their stability and responses to parameters of their chemical microenvironment.     

Calorimetric dissection of thermal unfolding of OspA,a predominantly beta-sheet protein containing a single-layer beta-sheet

Outer surface protein A (OspA) from Borrelia burgdorferi is a predominantly beta-sheet protein comprised of beta-strands beta1-beta21 and a short C-terminal alpha-helix. It contains two globular domains (N and C-terminal domains) and a unique single-layer beta-sheet (central beta-sheet) that connects the two domains. OspA contains an unusually large number of charged amino acid residues. To understand the mechanism of stabilization of this unique beta-sheet protein, thorough thermodynamic investigations of OspA and its truncated mutant lacking a part of the C-terminal domain were conducted using calorimetry and circular dichroism. The stability of OspA was found to be sensitive to pH and salt concentration. The heat capacity curve clearly consisted of two components, and all the thermodynamic parameters were obtained for each step. The thermodynamic parameters associated with the two transitions are consistent with a previously proposed model, in which the first transition corresponds to the unfolding of the C-terminal domain and the last two beta-strands of the central beta-sheet, and the second transition corresponds to that of the N-terminal domain and the first beta-strand of the central beta-sheet in the second peak. The ratio of calorimetric and van't Hoff enthalpies indicates that the first peak includes another thermodynamic intermediate state. Large heat capacity changes were observed for both transitions, indicative of large changes in the exposure of hydrophobic surfaces associated with the transitions. This observation demonstrates that hydrophobic parts are buried efficiently in the native structure in spite of the low content of hydrophobic residues in OspA. By decomposing the enthalpy, entropy, and Gibbs free energy into contributions from different interactions, we found that the enthalpy changes for hydrogen bonding and polar interactions are exceptionally large, indicating that OspA maintains its stability by making full use of its unique beta-sheet and high content of polar residues. These thermodynamic analyses demonstrated that it is possible to maintain protein tertiary structure by making effective use of an unusual amino acid composition.     

Structural energetics of the molten globule state

Certain partly ordered protein conformations, commonly called “moltenglobule states,” are widely believed to represent protein folding intermediates. Recentstructural studies of molten globule states ofdifferent proteins have revealed features whichappear to be general in scope. The emergingconsensus is that these partly ordered forms exhibit a high content of secondary structure, considerable compactness, nonspecific tertiary structure, and significant structural flexibility. These characteristics may be used to define ageneral state of protein folding called “the molten globule state,” which is structurally andthermodynamically distinct from both the native state and the denatured state. Despite exaatensive knowledge of structural features of afew molten globule states, a cogent thermodynamic argument for their stability has not yetbeen advanced. The prevailing opinion of thelast decade was that there is little or no enthalpy difference or heat capacity differencebetween the molten globule state and the unfolded state. This view, however, appears to beat variance with the existing database of protein structural energetics and with recent estimates of the energetics of denaturation of α-lactalbumin, cytochrome c, apomyoglobin, and T4 lysozyme. We discuss these four proteins at length. The results of structural studies, together with the existing thermodynamic values for fundamental interactions in proteins, provide the foundation for a structural thermodynamic framework which can account for the observed behavior of molten globule states. Within this framework, we analyze the physical basis for both the high stability of several molten globule states and the low probability of other protential folding intermediates. Additionally, we consider, in terms of reduced enthalpy changes and disrupted cooperative interactions, the thermodynamic basis for the apparent absence of a thermally induced, cooperative unfolding transition for some molten globule states. © 1993 Wiley-Liss, Inc.     

One-state downhill versus conventional protein folding

Classical protein folding invokes a cooperative transition between distinct thermodynamic states that are individually populated at equilibrium and separated by an energy barrier. It has been proposed, however, that the small protein, BBL, undergoes one-step downhill folding whereby it folds non-cooperatively to its native state without encountering an appreciable energy barrier. Only a single conformational ensemble is populated under given conditions, and so the denatured state ensemble progressively changes into the native structure. A wide dispersion of thermal denaturation midpoints that was observed for an extrinsically labelled fragment of BBL is proposed to be evidence for its one-state, downhill folding, a phenomenon that is also suggested to be functionally important for BBL and its homologues. We found, however, that thermal denaturation of unlabelled wild-type BBL was highly cooperative, with very similar transition midpoints for the melting of secondary and tertiary interactions, as well as for individual residues when monitored by NMR. Similar results were also observed for two other homologues, E3BD and POB. Further, the extrinsic fluorophores perturbed the unfolding energetics of labelled BBL, and complicated its equilibrium behaviour. One-step downhill folding may well occur for some proteins that do not have distinct folded states but not for BBL and its well-folded homologues.