Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.     

Kinetics of folding nuclei formation in proteins

When a protein folds or unfolds, it passes through many half-folded microstates. Only a few of them can accumulate and be seen experimentally, and this happens only when the folding (or unfolding) occurs far from the point of thermodynamic equilibrium between the native and denatured states. The universal features of folding, though, are observed in the vicinity of the equilibrium point. Here the "two-state" transition proceeds without any accumulation of metastable intermediates, and only the transition state ("folding nucleus") is outlined by its key influence on the folding/unfolding kinetics. This review covers recent experimental and theoretical studies of folding nuclei.     

Folding Nuclei in Proteins

When a protein folds or unfolds, it passes through many half-folded microstates. Only a few of them can accumulate and be seen experimentally, and this happens only when the folding (or unfolding) occurs far from the point of thermodynamic equilibrium between the native and denatured states. The universal features of folding, though, are observed in the vicinity of the equilibrium point. Here the two-state transition proceeds without any accumulation of metastable intermediates, and only the transition state (folding nucleus) is outlined by its key influence on the folding/unfolding kinetics. This review covers recent experimental and theoretical studies of folding nuclei.     

An expanded view of the protein folding landscape of PDZ domains

Most protein domains fold in an apparently co-operative and two-state manner with only the native and denatured states significantly populated at any experimental condition. However, the protein folding energy landscape is often rugged and different transition states may be rate limiting for the folding reaction under different conditions, as seen for the PDZ protein domain family. We have here analyzed the folding kinetics of two PDZ domains and found that a previously undetected third transition state is rate limiting under conditions that stabilize the native state relative to the denatured state. In light of these results, we have re-analyzed previous folding data on PDZ domains and present a unified folding mechanism with three distinct transition states separated by two high-energy intermediates. Our data show that sequence composition tunes the relative stabilities of folding transition states within the PDZ family, while the overall mechanism is determined by topology. This model captures the kinetic folding mechanism of all PDZ domains studied to date.     

Folding nuclei in proteins

When a protein folds or unfolds, it passes through many half-folded microstates. Only a few of them can accumulate and be seen experimentally, and this happens only when the folding (or unfolding) occurs far from the point of thermodynamic equilibrium between the native and denatured states. The universal features of folding, though, are observed just close to the equilibrium point. Here the 'two-state' transition proceeds without any accumulation of metastable intermediates, and only the transition state ('folding nucleus') is outlined by its key influence on the folding-unfolding kinetics. Our aim is to review recent experimental and theoretical studies of the folding nuclei.     

High-pressure NMR spectroscopy for characterizing folding intermediates and denatured states of proteins

Extensive structural studies using high-pressure NMR spectroscopy have recently been carried out on proteins, which potentially contribute to our understanding of the mechanisms of protein folding. Pressure shifts the conformational equilibrium from higher to lower volume conformers. If the pressure is varied, starting from the folded native structure, in many cases we observe intermediate conformers before the onset of total unfolding. This enables the investigation of details of the structure and thermodynamic characteristics of various intermediate conformers of proteins under equilibrium conditions. We can also examine pressure effects on the structure and stability of some typical denatured states such as helical denatured, molten globule, and unfolded states. The high-pressure NMR method can also be used to investigate association/dissociation equilibria of oligomeric or aggregated proteins. Beside direct observation of kinetic intermediates upon pressure jump, NMR structural investigations of equilibrium conformers under pressure provide information about the structures of kinetic intermediates during folding/unfolding reactions.     

Alternate succession of steps can lead to the folding of a multidomain oligomeric protein

The beta 2 subunit of Escherichia coli tryptophan synthase can be either unfolded in 6 M guanidine, or extensively denatured at acidic pH. These two denatured forms of beta 2 have different circular dichroism spectra and thus correspond to distinct physical states. Here we compare the folding pathways of these two different denatured forms of beta chains. We describe the kinetics of regain of a variety of physical, functional, and immunochemical signals characteristic of six successive steps previously identified on the folding pathway of guanidine unfolded beta 2. It is shown that whereas identical molecular events occur with the same kinetics, the two folding pathways are different, and involve different structural intermediates.     

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.     

Folding initiation sites and protein folding

The paper discusses the role of local structural preferences of protein segments in the folding of proteins. First a short overview of the local, secondary structures detected in peptides, protein fragments, denatured proteins and early folding intermediates is given. Next the discussion of their role in protein folding is presented based on recent literature and data obtained in our laboratory. In conclusion it is pointed out that, during folding, local structures populated at low levels in denatured state may facilitate the crossing of the folding transition state barrier, and consequently accelerate the rate limiting step in folding. However, the data show that this effect does not follow simple rules.     

Comparison of NMR structural and dynamics features of the urea and guanidine-denatured states of GED

Denatured states of proteins, the starting points as well as the intermediates of folding in vivo, play important roles in biological function. In this context, we describe here urea unfolding and characterization of the denatured state of GTPase effector domain (GED) of dynamin created by 9.7 M urea. These are compared with similar data for guanidine induced denaturation reported earlier. The unfolding characteristics in the two cases, as measured by the optical probes, are significantly different, urea unfolding proceeding via an intermediate. The structural and motional characteristics, determined by NMR, of the two denatured states are also strikingly different. The urea-denatured state shows a combination of α- and β-preferences in contrast to the entirely β-preferences in the guanidine-denatured state. Higher ¹⁵N transverse relaxation rates suggest higher folding propensities in the urea-denatured state. The implications of these to GED folding are discussed.     

Structural dynamics, stability and folding of proteins

The present concepts of protein folding in vitro are reviewed. According to these concepts, amino acid sequence of protein, which has appeared a result of evolutionary selection, determines the native structure of protein, the pathway of protein folding, and the existence of free energy barrier between native and denatured states of protein. The latter means that protein macromolecule can exist in either native or denatured state. And all macromolecules in the native state are identical but for structural fluctuations due to Brownian motion of their atoms. Identity of all molecules in native state is of primary importance for their correct functioning. The dependence of protein stability, which is measured as the difference between free energy of protein in native and denatured states, on temperature and denaturant concentration is discussed. The modern approaches characterizing transition state and nucleation are regarded. The role of intermediate and misfolded states in amorphous aggregate and amyloid fibril formation is discussed.     

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.     

The denatured state of Engrailed Homeodomain under denaturing and native conditions

Protein folding starts from the elusive form of the denatured state that is present under conditions that favour the native state. We have studied the denatured state of Engrailed Homeodomain (En-HD) under mildly and strongly denaturing conditions at the level of individual residues by NMR and more globally by conventional spectroscopy and solution X-ray scattering. We have compared these states with a destabilized mutant, L16A, which is predominantly denatured under conditions where the wild-type is native. This engineered denatured state, which could be directly studied under native conditions, was in genuine equilibrium with the native state, which could be observably populated by changing the conditions or introducing a stabilizing mutation. The denatured state had extensive native secondary structure and was significantly compact and globular. But, the side-chains and backbone were highly mobile. Non-cooperative melting of the residual structure on the denatured state of En-HD was observed, both at the residue and the molecular level, with increasingly denaturing conditions. The absence of a co-operative transition could result from the denatured state ensemble progressing through a series of intermediates or from a more general slide (second-order transition) from the compact form under native conditions to the more extended at highly denaturing conditions. In either case, the starting point for folding under native conditions is highly structured and already poised to adopt the native structure.     

Conformational heterogeneity of an equilibrium folding intermediate quantified and mapped by scanning mutagenesis

It is challenging to experimentally define an energy landscape for protein folding that comprises multiple partially unfolded states. Experimental results are often ambiguous as to whether a non-native state is conformationally homogeneous. Here, we tested an approach combining systematic mutagenesis and a Br?nsted-like analysis to reveal and quantify conformational heterogeneity of folding intermediate states. Using this method, we resolved an otherwise apparently homogeneous equilibrium folding intermediate of Borrelia burgdorferi OspA into two conformationally distinct species and determined their relative populations. Furthermore, we mapped the structural differences between these intermediate species, which are consistent with the non-native species that we previously proposed based on native-state hydrogen exchange studies. When treated as a single state, the intermediate ensemble exhibited fractional Phi-values for mutations and Hammond-type behaviors that are often observed for folding transition states. We found that a change in relative population of the two species within the intermediate ensemble explains these properties well, suggesting that fractional Phi-values and Hammond-type behaviors exhibited by folding intermediates and transition states may arise more often from conformational heterogeneity than from a single partial structure. Our results are consistent with the presence of multiple minima in a rugged energy landscape predicted from theoretical studies. The method described here provides a promising means to probe a complex folding energy landscape.     

Formation of native and non-native interactions in ensembles of denatured ACBP molecules from paramagnetic relaxation enhancement studies

Paramagnetic relaxation enhancement measurements in the denatured state of ACBP have provided distance restraints that have been used in computer simulations to determine the conformational ensembles representing the denatured states of ACBP under a variety of conditions. A detailed comparison of the residual structure in the denatured state of ACBP under these different conditions has enabled us to infer that regions in the N and C-terminal parts of the protein sequence have a high tendency to interact in the unfolded state under physiological conditions. By comparing the structural features in the denatured states with those in the transition state for folding we also provided new insights into the mechanism of formation of the native state of this protein.     

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.     

Folding pathway of FKBP12 and characterisation of the transition state.

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast "burst" phases are observed. FKBP12, with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (kF=4 s(-1)at 25 degrees C). The topological crossing of two loops in FKBP12, a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway.The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. In addition, unfolding and refolding studies in the presence of ligand provided information on the structure of the ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70 % of the surface area buried. The ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the alpha-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper).     

Morphogenesis of a protein: folding pathways and the energy landscape

Current knowledge on the reaction whereby a protein acquires its native three-dimensional structure was obtained by and large through characterization of the folding mechanism of simple systems. Given the multiplicity of amino acid sequences and unique folds, it is not so easy, however, to draw general rules by comparing folding pathways of different proteins. In fact, quantitative comparison may be jeopardized not only because of the vast repertoire of sequences but also in view of a multiplicity of structures of the native and denatured states. We have tackled the problem of the relationships between the sequence information and the folding pathway of a protein, using a combination of kinetics, protein engineering and computational methods, applied to relatively simple systems. Our strategy has been to investigate the folding mechanism determinants using two complementary approaches, i.e. (i) the study of members of the same family characterized by a common fold, but substantial differences in amino acid sequence, or (ii) heteromorphic pairs characterized by largely identical sequences but with different folds. We discuss some recent data on protein-folding mechanisms by presenting experiments on different members of the PDZ domain family and their circularly permuted variants. Characterization of the energetics and structures of intermediates and TSs (transition states), obtained by Φ-value analysis and restrained MD (molecular dynamics) simulations, provides a glimpse of the malleability of the dynamic states and of the role of the topology of the native states and of the denatured states in dictating folding and misfolding pathways.