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.     

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.     

Effect of solvent conditions upon refolding pathways and intermediates for a simple lattice protein

Folding pathways and intermediates for a two-dimensional lattice protein have been investigated via computer simulation at various denaturant concentrations. The protein is represented as a chain of 8 hydrophobic (H) and 12 polar (P) beads on a square lattice sequenced in such a way that the native state is a compact hydrophobic core surrounded by a shell of polar beads. Two nonbonded H beads are said to attract each other with a potential of mean force of strength ϵ. Increasing |ϵ/kT| mimics decreasing the denaturant concentration in the solution. Dynamic Monte Carlo simulations have been performed in order to investigate the folding transition and the folding pathways. Sharp folding—unfolding transitions are observed and the folding process proceeds along well-defined pathways that are populated by partially folded intermediates. The folding pathways as well as the populations of the intermediates are strongly dependent upon the denaturant concentration. Generally, intermediates containing long open stretches of H beads are more populated at high denaturant concentration, whereas compact intermediates containing a substantial number of hydrophobic contacts are more populated at low denaturant concentrations. The folding process is also observed to be cooperative in nature in that the chain does not start folding until a key fold in the middle section of the chain is formed correctly. © 1997 John Wiley & Sons, Inc. Biopoly 42: 399–409, 1997     

Expanding the pressure technique: insights into protein folding from combined use of pressure and chemical denaturants

The fundamental principles derived from in vitro protein folding experiments have practical application in understanding the pathology of diseases of protein misfolding and for the development of industrial processes to produce proteins as pharmaceuticals and biotechnological reagents. High pressure as a tool to denature or disaggregate proteins offers a number of unique advantages. The emphasis of this review is on how low concentrations of chemical denaturants can be used in combination with high pressure to extend the range and scope of this useful technique. This approach has already been used in a number of studies, which are discussed here in the context of the questions they address. These include: the origin of the volume change observed on protein unfolding, pressure-induced formation of partially structured intermediates, pressure-induced dissociation of oligomeric and aggregated proteins, and the use of volume changes to probe the structure of the transition state. Wider use of hydrostatic pressure as a denaturation tool, facilitated by combination with chemical denaturants, is likely to bring significant advances to our understanding of protein structure, stability and folding, particularly in relation to proteins associated with the amyloid and prion diseases.     

The effect of disease-associated mutations on the folding pathway of human prion protein

Propagation of transmissible spongiform encephalopathies is believed to involve the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). An important step toward understanding the mechanism of this conversion is to elucidate the folding pathway(s) of the prion protein. We reported recently (Apetri, A. C., and Surewicz, W. K. (2002) J. Biol. Chem. 277, 44589-44592) that the folding of wild-type prion protein can best be described by a three-state sequential model involving a partially folded intermediate. Here we have performed kinetic stopped-flow studies for a number of recombinant prion protein variants carrying mutations associated with familial forms of prion disease. Analysis of kinetic data clearly demonstrates the presence of partially structured intermediates on the refolding pathway of each PrP variant studied. In each case, the partially folded state is at least one order of magnitude more populated than the fully unfolded state. The present study also reveals that, for the majority of PrP variants tested, mutations linked to familial prion diseases result in a pronounced increase in the thermodynamic stability, and thus the population, of the folding intermediate. These data strongly suggest that partially structured intermediates of PrP may play a crucial role in prion protein conversion, serving as direct precursors of the pathogenic PrP(Sc) isoform.     

New insights into the mechanisms of protein misfolding and aggregation in amyloidogenic diseases derived from pressure studies

Hydrostatic pressure is a robust tool for studying the thermodynamics of protein folding and protein interactions, as well as the dynamics and structure of folding intermediates. One of the main innovations obtained from using high pressure is the stabilization of folding intermediates such as molten-globule conformations, thus providing a unique opportunity for characterizing their structure and dynamics. Equally important is the prospect of understanding protein misfolding diseases by using pressure to populate partially folded intermediates at the junction between productive and off-pathway folding, which may give rise to misfolded proteins, aggregates, and amyloids. High hydrostatic pressure (HHP) has also been used to dissociate nonamyloid aggregates and inclusion bodies. In many proteins, the competition between correct folding and misfolding can lead to formation of insoluble aggregates, an important problem for the biotechnology industry and for human pathologies such as amyloidosis, Alzheimer's, Parkinson's, prion's, and tumor diseases. The diversity of diseases that result from protein misfolding has made this theme an important research focus for pharmaceutical and biotechnology companies. The use of high-pressure promises to contribute to the identification of the mechanisms behind these defects and creation of therapies against these diseases.     

Equilibrium collapse and the kinetic 'foldability' of proteins.

An important element of protein folding theory has been the identification of equilibrium parameters that might uniquely distinguish rapidly folding polypeptide sequences from those that fold slowly. One such parameter, termed sigma, is a dimensionless, equilibrium measure of the coincidence of chain compaction and folding that is predicted to be an important determinant of relative folding kinetics. To test this prediction and improve our understanding of the putative relationship between nonspecific compaction of the unfolded state and protein folding kinetics, we have used small-angle X-ray scattering and circular dichroism spectroscopy to measure the sigma of five well-characterized proteins. Consistent with theoretical predictions, we find that near-perfect coincidence of the unfolded state contraction and folding (sigma approximately 0) is associated with the rapid kinetics of these naturally occurring proteins. We do not, however, observe any significant correlation between sigma and either the relative folding rates of these proteins or the presence or absence of well-populated kinetic intermediates. Thus, while sigma approximately 0 may be a necessary condition to ensure rapid folding, differences in sigma do not account for the wide range of rates and mechanisms with which naturally occurring proteins fold.     

Energetics of protein structure and folding

The available experimental date on the kinetics of unfolding and refolding of small proteins are reviewed. Excluding slow transitions in the unfolded protein due to cis–trans isomerization of peptide bonds, the rate-limiting transition state in both unfolding and refolding is concluded to be a high-energy distortion of the fully folded state. Partially folded intermediates are undoubtedly important for folding, but their formation is normally not rate limiting. A simple model is used to illustrate some of the aspects of protein-folding energetics.     

Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants

Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the surfactant‐robust broad‐specificity proteases Savinase and Alcalase. Cleavage sites are identified by SDS‐PAGE and N‐terminal sequencing. We observe well‐defined cleavage fragments, which suggest that flexibility is limited to certain regions of the protein. Cleavage sites for α‐lactalbumin and myoglobin correspond to regions identified in other studies as partially unfolded at low pH or in the presence of organic solvents. For Tnfn3, which does not form partially folded structures under other conditions, cleavage sites can be rationalized from the structure of the protein's folding transition state and the position of loops in the native state. Nevertheless, they are more sensitive to choice of surfactant and protease, probably reflecting a heterogeneous and fluctuating ensemble of partially unfolded structures. Thus, for proteins accumulating stable intermediates on the folding pathway, surfactants encourage the formation of these states, while the situation is more complex for proteins that do not form these intermediates. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 221–231, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Fully oxidized scrambled isomers are essential and predominant folding intermediates of cardiotoxin-III

Scrambled isomers (X-isomers) are fully oxidized, non-native isomers of disulfide proteins. They have been shown to represent important intermediates along the pathway of oxidative folding of numerous disulfide proteins. A simple method to assess whether X-isomers present as folding intermediate is to conduct oxidative folding of fully reduced protein in the alkaline buffer alone without any supplementing thiol catalyst or redox agent. Cardiotoxin-III (CTX-III) contains 60 amino acids and four disulfide bonds. The mechanism of oxidative folding of CTX-III has been systematically characterized here by analysis of the acid trapped folding intermediates. Folding of CTX-III was shown to proceed sequentially through 1-disulfide, 2-disulfide, 3-disulfide and 4-disulfide (scrambled) isomers as folding intermediates to reach the native structure. When folding of CTX-III was performed in the buffer alone, more than 97% of the protein was trapped as 4-disulfide X-isomers, unable to convert to the native structure due to the absence of thiol catalyst. In the presence of thiol catalyst (GSH) or redox agents (GSH/GSSG), the recovery of native CTX-III was 80-85%. These results demonstrate that X-isomers play an essential and predominant role in the oxidative folding of CTX-III.     

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.     

Molecular basis of co-operativity in protein folding. III. Structural identification of cooperative folding units and folding intermediates.

The hierarchical partition function formalism for protein folding developed earlier has been extended through the use of three-dimensional polar and apolar contact plots. For each amino acid residue in the protein, these plots indicate the apolar and polar surfaces that are buried from the solvent, the identity of all amino acid residues that contribute to this shielding, and the magnitude of their contributions. These contact plots are then used to examine the distribution of the free energy of stabilization throughout the protein molecule. Analysis of these data allows identification of co-operative folding units and their hierarchical levels, and the identification of partially folded intermediates with a significant probability of being populated. The overall folding/unfolding thermodynamics of 12 globular proteins, for which crystallographic and experimental thermodynamics are available, is predicted within error. An energetic classification of partially folded intermediates is presented and the results compared to those cases for which structural and thermodynamic experimental information is available. Four different types of partially folded states and their structural energies are considered. (1) Local intermediates, in which only a local region of the protein loses secondary and tertiary interactions, while the rest of the protein remains intact. (2) Global intermediates, corresponding to the standard molten globule definition, in which significant secondary structure is maintained but native-like tertiary structure contacts are disrupted. (3) Extended intermediates characterized by the existence of secondary structure elements (e.g. alpha-helices) exposed to solvent. (4) Folding intermediates in proteins with two structural domains. The structure and energetics of folding intermediates of apo-myoglobin, alpha-lactalbumin, phosphoglycerate kinase and arabinose-binding protein are considered in detail.     

Conformation and thermodynamic stability of pre-molten and molten globule states of mammalian cytochromes-c

Proteins in cells fold via a number of intermediates. These intermediates are quite important as they guide the protein to attain its unique native conformation. To solve the immensely difficult problem of protein folding, it is necessary to characterize intermediates which will unravel the mystery of the steps involved in the proper folding of proteins. Cytochromes-c (cyts-c) have played an important role in studies of the earliest events and intermediates in protein folding. They have always been considered as model proteins for protein folding studies due to their intrinsic properties that can be measured by multiple probes. A large number of different solvent conditions have been employed to obtain equilibrium intermediates of cyts-c. These intermediates show structural heterogeneity which is mainly due to the different solvent conditions used to induce them. In this review we present results of conformational and thermodynamic characterization of equilibrium intermediates (molten globules and pre-molten globules) of the mammalian cyts-c under different solvent conditions.     

Lipid dependant disorder-to-order conformational transitions in apolipoprotein CI derived peptides

In contrast to the notion established for many years that protein function depends on rigid 3D structures, nowadays there is important evidence suggesting that non-structured segments of proteins play important roles in protein function. Therefore, disorder-to-order dynamic conformational transitions have been proposed as an attractive mechanism involved in protein-protein recognition. Our laboratory using Langmuir monolayers of apolipoproteins has previously shown that upon lateral compression at the air/water and phospholipid/water interfaces, there is an important movement of the C-terminal segment of apolipoprotein CI towards the air, considered the hydrophobic region of the monolayer and the acyl-chain region of the interface when phospholipids are used. Here, in an attempt to define secondary structure changes that might occur within this C-terminal segment of apoCI while moving from the monolayer interface back and forth its hydrophobic region, employing three peptides derived from apoCI we studied by circular dichroism and dynamic light scattering their conformational properties when associated to a series of amphipathic lipids and lipid-like molecules. Our results show that a series of lysophospholipids present the ability to modulate the formation of an α helix at the C-terminal peptide of apoCI through a disorder-to-order transition while forming small lipid/peptide aggregates below 10 nm in diameter.     

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.     

Reversible unfolding and refolding behavior of a monomeric aldolase from Staphylococcus aureus.

Thermal and GdmCl-induced unfolding transitions of aldolase from Staphylococcus aureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a delta G0 value of -9 +/- 2 kJ/mol at 20 degrees C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 degrees C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of alpha/beta barrel proteins.     

A novel method to determine thermal transition curves of disulfide-containing proteins and their structured folding intermediates

The stability of a protein or of its folding intermediates is frequently characterized by its resistance to chemical and/or thermal denaturation. The folding/unfolding process is generally followed by spectroscopic methods such as absorbance, fluorescence, circular dichroism spectroscopy, etc. Here, we demonstrate a new method, by using HPLC, for determining the thermal unfolding transitions of disulfide-containing proteins and their structured folding intermediates. The thermal transitions of a model protein, ribonuclease A (RNase A), and a recently found unfolding intermediate of onconase (ONC), des [30-75], have been estimated by this method. Finally, the advantages of this method over traditional techniques are discussed by providing specific examples.     

Autonomous subdomains in protein folding.

Proteolytic dissection of native trp repressor and horse heart cytochrome c has been used to infer some of the steps in the folding pathways of the intact proteins. For both proteins, small fragments are capable of undergoing spontaneous noncovalent association to form subdomains with native-like secondary and/or tertiary structural features, suggesting that dissection/reassembly may be a general method to gain insight into the structures of folding intermediates. The importance of this approach is its simplicity and potential applicability to studying the folding pathways of a wide range of proteins. The proteases report on the structure and dynamics of the native state, circumventing the need for prior knowledge of the structures of folding intermediates. The observation that small fragments of proteins can associated noncovalently suggests that protein folding can be viewed as an intramolecular "recognition" process. The results imply that substantial information about protein structure and folding is encoded at the level of subdomains, and that chain connectivity has only a minor role in determining the fold.     

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.     

Theory of protein folding

Protein folding should be complex. Proteins organize themselves into specific three-dimensional structures, through a myriad of conformational changes. The classical view of protein folding describes this process as a nearly sequential series of discrete intermediates. In contrast, the energy landscape theory of folding considers folding as the progressive organization of an ensemble of partially folded structures through which the protein passes on its way to the natively folded structure. As a result of evolution, proteins have a rugged funnel-like landscape biased toward the native structure. Connecting theory and simulations of minimalist models with experiments has completely revolutionized our understanding of the underlying mechanisms that control protein folding.