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.     

Compact dimension of denatured states of staphylococcal nuclease

Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.     

Noninteracting local-structure model of folding and unfolding transition in globular proteins. I. Formulation

A statistical-mechanical model (a noninteracting local structure model) of folding and unfolding transition in globular proteins is described and a formulation is given to calculate the partition function. The process of transition is discussed in this model within the framework of equilibrium statistical mechanics. In order to clarify the range of applicability of such an approach, the characteristics of the folding and unfolding transition in globular proteins are analyzed from the statistical-physical point of view. A theoretical advantage is pointed out in studying folding and unfolding processes taking place as conformational fluctuations in individual protein molecules under macroscopic equilibrium at the melting temperature. In this case, paths of folding and unfolding are shown to be identical in the statistical sense. A key to the noninteracting local structure model lies in the concept of local structures and the assumption of the absence of interactions between local structures. A local structure is defined as a continuous section of the chain which takes the same or similar local conformation as in the native conformation. The assumption of the absence of inter-actions between local structures endows the model with the remarkable character that its partition function can be calculated exactly; thereby the equilibrium population of various conformations along the folding and unfolding paths can be discussed only by a knowledge of the folded native conformation.     

Mesophile versus thermophile: insights into the structural mechanisms of kinetic stability

Obtaining detailed knowledge of folding intermediate and transition state (TS) structures is critical for understanding protein folding mechanisms. Comparisons between proteins adapted to survive extreme temperatures with their mesophilic homologs are likely to provide valuable information on the interactions relevant to the unfolding transition. For kinetically stable proteins such as alpha-lytic protease (alphaLP) and its family members, their large free energy barrier to unfolding is central to their biological function. To gain new insights into the mechanisms that underlie kinetic stability, we have determined the structure and high temperature unfolding kinetics of a thermophilic homolog, Thermobifida fusca protease A (TFPA). These studies led to the identification of a specific structural element bridging the N and C-terminal domains of the protease (the "domain bridge") proposed to be associated with the enhanced high temperature kinetic stability in TFPA. Mutagenesis experiments exchanging the TFPA domain bridge into alphaLP validate this hypothesis and illustrate key structural details that contribute to TFPA's increased kinetic thermostability. These results lead to an updated model for the unfolding transition state structure for this important class of proteases in which domain bridge undocking and unfolding occurs at or before the TS. The domain bridge appears to be a structural element that can modulate the degree of kinetic stability of the different members of this class of proteases.     

Essential dynamics of reversible peptide folding: memory-free conformational dynamics governed by internal hydrogen bonds

A principal component analysis has been applied on equilibrium simulations of a beta-heptapeptide that shows reversible folding in a methanol solution. The analysis shows that the configurational space contains only three dense sub-states. These states of relatively low free energy correspond to the "native" left-handed helix, a partly helical intermediate, and a hairpin-like structure. The collection of unfolded conformations form a relatively diffuse cloud with little substructure. Internal hydrogen-bonding energies were found to correlate well with the degree of folding. The native helical structure folds from the N terminus; the transition from the major folding intermediate to the native helical structure involves the formation of the two most C-terminal backbone hydrogen bonds. A four-state Markov model was found to describe transition frequencies between the conformational states within error limits, indicating that memory-effects are negligible beyond the nanosecond time-scale. The dominant native state fluctuations were found to be very similar to unfolding motions, suggesting that unfolding pathways can be inferred from fluctuations in the native state. The low-dimensional essential subspace, describing 69% of the collective atomic fluctuations, was found to converge at time-scales of the order of one nanosecond at all temperatures investigated, whereas folding/unfolding takes place at significantly longer time-scales, even above the melting temperature.     

Understanding beta-hairpin formation by molecular dynamics simulations of unfolding

We have studied the mechanism of formation of a 16-residue beta-hairpin from the protein GB1 using molecular dynamics simulations in an aqueous environment. The analysis of unfolding trajectories at high temperatures suggests a refolding pathway consisting of several transient intermediates. The changes in the interaction energies of residues are related with the structural changes during the unfolding of the hairpin. The electrostatic energies of the residues in the turn region are found to be responsible for the transition between the folded state and the hydrophobic core state. The van der Waals interaction energies of the residues in the hydrophobic core reflect the behavior of the radius of gyration of the core region. We have examined the opposing influences of the protein-protein (PP) energy, which favors the native state, and the protein-solvent (PS) energy, which favors unfolding, in the formation of the beta-hairpin structure. It is found that the behavior of the electrostatic components of PP and PS energies reflects the structural changes associated with the loss of backbone hydrogen bonding. Relative changes in the PP and PS van der Waals interactions are related with the disruption of the hydrophobic core of a protein. The results of the simulations support the hydrophobic collapse mechanism of beta-hairpin folding.     

Long-range electrostatic effects on peptide folding.

The reversible folding/unfolding of a short peptide in solution is studied by molecular dynamics simulations. The effects of long-range electrostatic interactions are examined and found to be important both for the equilibrium between folded and unfolded states and the dynamics of the folding process. The neglect of long-range electrostatics leads to an increased population of unfolded states and increased structural fluctuations. When such interactions are taken into account, the peptide unfolds and folds to the experimentally determined structure several times during a 25 ns trajectory, with approximately equal populations of folded and unfolded states in the neighborhood of its proposed melting temperature. The effect of using spherical boundary conditions rather than periodic ones does not appear to have any major effect on the folding dynamics.     

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).     

The nature of folded states of globular proteins.

We suggest, using dynamical simulations of a simple heteropolymer modelling the alpha-carbon sequence in a protein, that generically the folded states of globular proteins correspond to statistically well-defined metastable states. This hypothesis, called the metastability hypothesis, states that there are several free energy minima separated by barriers of various heights such that the folded conformations of a polypeptide chain in each of the minima have similar structural characteristics but have different energies from one another. The calculated structural characteristics, such as bond angle and dihedral angle distribution functions, are assumed to arise from only those configurations belonging to a given minimum. The validity of this hypothesis is illustrated by simulations of a continuum model of a heteropolymer whose low temperature state is a well-defined beta-barrel structure. The simulations were done using a molecular dynamics algorithm (referred to as the "noisy" molecular dynamics method) containing both friction and noise terms. It is shown that for this model there are several distinct metastable minima in which the structural features are similar. Several new methods of analyzing fluctuations in structures belonging to two distinct minima are introduced. The most notable one is a dynamic measure of compactness that can in principle provide the time required for maximal compactness to be achieved. The analysis shows that for a given metastable state in which the protein has a well-defined folded structure the transition to a state of higher compactness occurs very slowly, lending credence to the notion that the system encounters a late barrier in the process of folding to the most compact structure. The examination of the fluctuations in the structures near the unfolding----folding transition temperature indicates that the transition state for the unfolding to folding process occurs closer to the folded state.     

General dynamic properties of Abeta12-36 amyloid peptide involved in Alzheimer's disease from unfolding simulation

To study the folding/unfolding properties of a beta-amyloid peptide Abeta(12-36) of Alzheimer's disease, five molecular dynamics simulations of Abeta(12-36) in explicit water were done at 450 K starting from a structure that is stable in trifluoroethanol/water at room temperature with two alpha-helices. Due to high temperature, the initial helical structure unfolded during the simulation. The observed aspects of the unfolding were as follows. 1) One helix (helix 1) had a longer life than the other (helix 2), which correlates well with the theoretically computed Phi values. 2) Temporal prolongation of helix 1 was found before unfolding. 3) Hydrophobic cores formed frequently with rearrangement of amino-acid residues in the hydrophobic cores. The formation and rearrangement of the hydrophobic cores may be a general aspect of this peptide in the unfolded state, and the structural changes accompanied by the hydrophobic-core rearrangement may lead the peptide to the most stable structure. 4) Concerted motions (collective modes) appeared to unfold helix 1. The collective modes were similar with those observed in another simulation at 300 K. The analysis implies that the conformation moves according to the collective modes when the peptide is in the initial stage of protein unfolding and in the final stage of protein folding.     

Thermodynamic effects of mutations on the denaturation of T4 lysozyme.

We investigated the folding of substantially destabilized mutant forms of T4 lysozyme using differential scanning calorimetry and circular dichroism measurements. Three mutations in an alpha-helix in the protein's N-terminal region, the alanine insertion mutations S44[A] and K48[A], and the substitution A42K had previously been observed to result in unexpectedly low apparent enthalpy changes of melting, compared to a pseudo-wild-type reference protein. The pseudo-wild-type reference protein thermally unfolds in an essentially two-state manner. However, we found that the unfolding of the three mutant proteins has reduced cooperativity, which partially explains their lower apparent enthalpy changes. A three-state unfolding model including a discrete intermediate is necessary to describe the melting of the mutant proteins. The reduction in cooperativity must be considered for accurate calculation of the energy changes of folding. Unfolding in two stages reflects the underlying two-subdomain structure of the lysozyme protein family.     

Disulfide bonds and protein folding

The applications of disulfide-bond chemistry to studies of protein folding, structure, and stability are reviewed and illustrated with bovine pancreatic ribonuclease A (RNase A). After surveying the general properties and advantages of disulfide-bond studies, we illustrate the mechanism of reductive unfolding with RNase A, and discuss its application to probing structural fluctuations in folded proteins. The oxidative folding of RNase A is then described, focusing on the role of structure formation in the regeneration of the native disulfide bonds. The development of structure and conformational order in the disulfide intermediates during oxidative folding is characterized. Partially folded disulfide species are not observed, indicating that disulfide-coupled folding is highly cooperative. Contrary to the predictions of "rugged funnel" models of protein folding, misfolded disulfide species are also not observed despite the potentially stabilizing effect of many nonnative disulfide bonds. The mechanism of regenerating the native disulfide bonds suggests an analogous scenario for conformational folding. Finally, engineered covalent cross-links may be used to assay for the association of protein segments in the folding transition state, as illustrated with RNase A.     

Characterisation of denatured states of sensory rhodopsin II by solution-state NMR

Sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis, is a novel model system for membrane protein folding studies. Recently, the SDS-denatured states and the kinetics for reversible unfolding of pSRII have been investigated, opening the door to the first detailed characterisation of denatured states of a membrane protein by solution-state nuclear magnetic resonance (NMR) using uniformly ¹⁵N-labelled pSRII. SDS denaturation and acid denaturation of pSRII both lead to fraying of helix ends but otherwise small structural changes in the transmembrane domain, consistent with little changes in secondary structure and disruption of the retinal-binding pocket and tertiary structure. Widespread changes in the backbone amide dynamics are detected in the form of line broadening, indicative of μs-to-ms timescale conformational exchange in the transmembrane region. Detailed analysis of chemical shift and intensity changes lead to high-resolution molecular insights on structural and dynamics changes in SDS- and acid-denatured pSRII, thus highlighting differences in the unfolding pathways under the two different denaturing conditions. These results will form the foundation for furthering our understanding on the folding and unfolding pathways of retinal-binding proteins and membrane proteins in general, and also for investigating the importance of ligand-binding in the folding pathways of other ligand-binding membrane proteins, such as GPCRs.     

Phage P22 tailspike protein: removal of head-binding domain unmasks effects of folding mutations on native-state thermal stability.

A shortened, recombinant protein comprising residues 109-666 of the tailspike endorhamnosidase of Salmonella phage P22 was purified from Escherichia coli and crystallized. Like the full-length tailspike, the protein lacking the amino-terminal head-binding domain is an SDS-resistant, thermostable trimer. Its fluorescence and circular dichroism spectra indicate native structure. Oligosaccharide binding and endoglycosidase activities of both proteins are identical. A number of tailspike folding mutants have been obtained previously in a genetic approach to protein folding. Two temperature-sensitive-folding (tsf) mutations and the four known global second-site suppressor (su) mutations were introduced into the shortened protein and found to reduce or increase folding yields at high temperature. The mutational effects on folding yields and subunit folding kinetics parallel those observed with the full-length protein. They mirror the in vivo phenotypes and are consistent with the substitutions altering the stability of thermolabile folding intermediates. Because full-length and shortened tailspikes aggregate upon thermal denaturation, and their denaturant-induced unfolding displays hysteresis, kinetics of thermal unfolding were measured to assess the stability of the native proteins. Unfolding of the shortened wild-type protein in the presence of 2% SDS at 71 degrees C occurs at a rate of 9.2 x 10(-4) s(-1). It reflects the second kinetic phase of unfolding of the full-length protein. All six mutations were found to affect the thermal stability of the native protein. Both tsf mutations accelerate thermal unfolding about 10-fold. Two of the su mutations retard thermal unfolding up to 5-fold, while the remaining two mutations accelerate unfolding up to 5-fold. The mutational effects can be rationalized on the background of the recently determined crystal structure of the protein.     

Kinetics of the helix/coil transition of the collagen-like peptide (Pro-Hyp-Gly)10

This article measures the rates of folding and unfolding of the collagen-like peptide (Pro-Hyp-Gly)(10) over overlapping concentration and temperature ranges. The data allow calculation of the orders of the folding and the unfolding reactions, the effective Arrhenius activation energies, and numerical solution of the differential equation controlling the helix/coil transition during temperature scanning. The resulting predictions of helicity closely followed DSC measurements of the peptide in both up- and down-scanning modes, confirming the validity of the theoretical equations governing the kinetics of the folding/unfolding process. In both up- and down-scanning, three regions were apparent: "quasistatic," "rate," and "mixed." At very low scanning rates, a quasistatic region revealed a broad, short endotherm that was independent of scanning rate, but dependent on concentration and equal to the equilibrium endotherm. At high up-scanning rates, the "rate region" endotherm was sharp and tall and T(max) increased with scanning rate. In down-scanning, the "rate peak" was very broad and very short and T(max) decreased with scanning rate. The "mixed region" showed nascent "rate" and nascent "quasistatic" peaks, which were evident in the same up-scan under certain conditions. Comparison of (Pro-Hyp-Gly)(10) and (Pro-Pro-Gly)(10) showed that the higher temperature stability of (Pro-Hyp-Gly)(10) is due mainly to its slower rate of unfolding and higher activation energy.     

Structural Characterization of the Cooperativity of Unfolding of a Heterodimeric Protein using Hydrogen Exchange-Mass Spectrometry

Little is known about how the sequence of structural changes in one chain of a heterodimeric protein is coupled to those in the other chain during protein folding and unfolding reactions, and whether individual secondary structural changes in the two chains occur in one or many coordinated steps. Here, the unfolding mechanism of a small heterodimeric protein, double chain monellin, has been characterized using hydrogen exchange-mass spectrometry. Transient structure opening, which enables HX, was found to be describable by a five state N ↔ I₁ ↔ I₂ ↔ I₃ ↔ U mechanism. Structural changes occur gradually in the first three steps, and cooperatively in the last step. β strands 2, 4 and 5, as well as the α-helix undergo transient unfolding during all three non-cooperative steps, while β1 and the two loops on both sides of the helix undergo transient unfolding during the first two steps. In the absence of GdnHCl, only β3 in chain A of the protein unfolds during the last cooperative step, while in the presence of 1 M GdnHCl, not only β3, but also β2 in chain B unfolds cooperatively. Hence, the extent of cooperative structural change and size of the cooperative unfolding unit increase when the protein is destabilized by denaturant. The naturally evolved two-chain variant of monellin folds and unfolds in a more cooperative manner than does a single chain variant created artificially, suggesting that increasing folding cooperativity, even at the cost of decreasing stability, may be a driving force in the evolution of proteins.     

Molecular dynamics simulations of helix denaturation.

An understanding of the structural transitions that an alpha-helix undergoes will help to elucidate such motions in proteins and their role in protein folding. We present the results of molecular dynamics simulations to investigate these transitions in a short polyalanine peptide (13 residues) both in vacuo and in the presence of solvent. The denaturation of this peptide was monitored as a function of temperature (ranging from 5 to 200 degrees C). In vacuo, the helical state predominated at all temperatures, whereas in solution the helix melted with increasing temperature. The peptide was predominantly helical at low temperature in solution, while at intermediate temperatures the peptide spent the bulk of the time fluctuating between different conformations with intermediate amounts of helix, e.g. not completely helical nor entirely non-helical. Many of these conformations consisted of short helical segments with intervening non-helical residues. At high temperature the peptide unfolded and adopted various collapsed unstructured states. The intrahelical hydrogen bonds that break at high temperature were not fully compensated by hydrogen bonds with water molecules in the partially unfolded forms of the peptide. Increases in temperature disrupted both the helical structure and the peptide-water interactions. Water played a major but indirect role in facilitating unfolding, as opposed to specifically competing for the intrapeptide hydrogen bonds. The implications of our results to protein folding are discussed.     

Pathway-dependent refolding of E. coli 5S RNA.

The refolding of 5S RNA into its two conformational states has been examined as a function of solvent composition and annealing conditions. The results show that the product distribution depends on the folding pathway. Quick cooling from high temperature produces roughly equal amounts of the two forms, even in the presence of 1 mm Mg++. However annealing by slow cooling to intermediate temperatures (50 degrees--60 degrees C) in Mg++-containing buffers, followed by quick cooling, allows formation of a structure which guides the refolding path to the "native" conformation. The stability of this structural nucleus for the "native" conformation depends strongly on Mg++ concentration. We conclude that the A ("native") conformation differs from the B conformation not in rate of refolding, but rather in having a lower enthalpy and a also a smaller rate of unfolding for the critical structural nucleus. The order of folding during biosynthesis may be crucial for forming the "native" conformation.     

Effect of temperature on structure and functional properties of horseradish peroxidase

The dynamics of structural and functional changes proceeding in a peroxidase molecule under the effect of temperature was studied. It was shown that peroxidase thermoinactivation proceeds in two consequent stages. Based on the analysis of enzyme peroxidase and oxidase activity and peroxidase spectral and buffer characteristics, it was established that at temperatures from 20 to 55 degrees C reversible conformation there occur changes of the hemoprotein molecule related to consequent unfolding and folding of the protein globule. The influence of temperature of 60 degrees C and above induces the protein globule unfolding and loosing of peroxidase activity.     

Exploiting the Link between Protein Rigidity and Thermostability for Data‐Driven Protein Engineering

Understanding and exploiting the relationship between microscopic structure and macroscopic stability is important for developing strategies to improve protein stability at high temperatures. The thermostability of proteins has been repeatedly linked to an enhanced structural rigidity of the folded native state. In the current study, the rigidity of protein structures from mesophilic and thermophilic organisms along a thermal unfolding trajectory is directly probed. In order to perform this, protein structures were modeled as constraint networks, and the rigidity in these networks was quantified using the Floppy Inclusion and Rigid Substructure Topography (FIRST) method. During the thermal unfolding, a phase transition was observed that defines the rigidity percolation threshold and corresponds to the folded‐unfolded transition in protein folding. Using concepts from percolation theory and network science, a higher phase transition temperature was observed for ca. two‐thirds of the proteins from thermophilic organisms compared to their mesophilic counterparts, when applied to a data set of 20 pairs of homologues. From both the analysis of the microstructure of the constraint networks and monitoring the macroscopic behavior during the thermal unfolding, direct evidence was found for the “corresponding states” concept, which states that mesophilic and thermophilic enzymes are in corresponding states of similar flexibility at their respective optimal temperature. Finally, the current approach facilitated the identification of structural features from which a destabilization of the structure originates upon thermal unfolding. These predictions show a good agreement with the experimental data. Therefore, the information might be exploited in data‐driven protein engineering by pointing to residues that should be varied to obtain a protein with higher thermostability.