Collapse and cooperativity in protein folding

Collapse and cooperativity in protein folding Andrew D Miranker* and Christopher M Dobsont The folding of a polypeptide chain is associated both with compactness and cooperativity within local and global regions of the protein structure, and with the formation of the native-like molecular architecture. Recent experiments shed light on these issues and their relationships to the pathways of protein folding.

Address *t New Chemistry Laboratory, Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford OX1 3CIT, UK *e-mail: [email protected] t e-mail: [email protected] Current Opinion in Structural Biology 1996, 6:31-42 © Current Biology Ltd ISSN 0959-440X

Abbreviations ACBP acyl-coenzyme A binding protein ANS 1-anilinonaphthalenesulfonicacid CD circulardichroism CI2 chymotrypsin ilqhibitor 2 CspB cold-shockprotein from Bacillus subti/is DHFR dihydrofolate reductase

Introduction From experimental studies, it appears that the folding pathways of globular proteins are varied and complex [1°]. An underlying set of rules must, however, surely exist; it is most improbable that every case of protein folding represents a unique solution to the problem of protein structure formation. The native states of polypeptides are distinguishable from their unfolded forms by their high degree of compactness, the existence of a hydrophobic core and a hydtophilic surface, a well defined overall architecture and the presence of specific and cooperative interactions between buried side chains. The process of protein folding involves the generation of all these characteristics and recent progress in both instrumentation and experimental design has enabled unprecedented insights to be gained into how it happens. In this review, we focus on the highlights of studies carried out during the past year and, in particular, identify specific issues concerned with the role and character of compact non-native states which are emerging as key indicators of the nature of the underlying principles involved in protein folding. Some of these are illustrated schematically in Figure 1. The nature of pathways One of the principal means of studying folding is the detailed kinetic investigation of events following transfer of a protein from denaturing to non-denaturing conditions or vice versa. Under these non-equilibrium conditions, two types of kinetic behaviour have been found experimentally in studies of the folding of different

proteins. In the first, the formation of the native state, at least to a good approximation, follows two-state kinetics: no intermediates are detectable between the highly unfolded and the folded state. The necessary conditions of this behaviour are that all properties that characterize the development of structure follow the same monophasic kinetic curve. Furthermore, properties of the transition state characterized by kinetic analysis should be independent of the direction of the reaction. Such behaviour has been analyzed in great detail for chymotrypsin inhibitor 2 (CI2) and a variety of site-directed mutants 12]. Close agreement has been found between parameters calculated from kinetic folding and unfolding reactions and those derived from equilibrium studies. Similar behaviour has also been identified for a number of other proteins, all of which are small (<100 residues) and have well defined folds. Two recently studied proteins include a representative of all-et proteins, acyl-coenzyme A binding protein (ACBP) [3°], and a representative of all-13 proteins, cold-shock protein from Bacillus subtilis (CspB) [4°]. The simplicity of a two-state system makes it attractive for studies designed to probe the transition state of the folding process. The existence, multiplicity and character of transition states are key issues in the analysis of kinetic data. While kinetic observables are typically interpreted on the basis of a simple reaction coordinate with initial, final, intermediate and transition states, the high dimensionality, the interplay of entropic as well as enthalpic elements and, perhaps most importantly, the changing role of the solvent during folding make for a highly complex and as yet undefined reaction coordinate. One approach to addressing these issues is based on protein-engineering techniques and aims to give an order parameter for the localized comparison of the transition state with the native state [5°]. The parameter ~ is calculated as the change in the free energy difference between the transition state and the unfolded state relative to the change in the overall protein stability as a result of site-directed mutagenesis. In situations where a mutation has an identical energetic effect on the folded and transition states, the • value is 1 and the local structure is assumed to be identical. By contrast, in cases where ~ = 0 , the structure of the transition state in this region is assumed to be identical to that of the unfolded state. For CI2, the various residues studied appear to have fractional • values [6*], making interpretation of these resuits particularly difficult [7]. From many detailed studies, however, the transition state of CI2 has been interpreted as a globally collapsed state strongly resembling the folded protein but expanded by -30% and lacking the specific hydrophobic packing characteristics of the native state [5°]. This type of approach to defining a transition state is

32

Folding and binding

Figure 1

___ Denat ur!n_gcond i_ti_o_ns__ _ _ Native conditions -,

~ ",.

¢

~ , 4...

(b) -•J~-~,••"'"

)

F-"(c'i

(e)

c 1 9 9 6 Current Opinion in Structural Biology

Schematic representation of the folding of a simple protein. (a) In the unfolded state of a protein under highly denaturing conditions, a large number of conformational states are accessible and local structural preferences are limited primarily by steric restrictions and excluded volume effects. (b) When placed under refolding conditions, local interactions result in nascent secondary structure and/or nucleation sites, the distribution of which will be dependent on refotding conditions. The fraction of native contacts at this point is small, as is the degree of global cooperativity. The process of collapse is linked with the formation of largely native-like secondary structure. These events are principally driven by the intrinsic tendency of a heteropolymer in aqueous solution to form a hydrophobic core. The pattern of residue types (polar residues are represented as crosses and hydrophobic residues are represented as dots) is crucial to stabilizing secondary structure and dictating the overall architecture of the collapsed state. (g) The native protein in this example is represented as a right-handed up-down-up-down four-helix bundle. Within the collapsed state, various structural families might exist. Some of these may have incorrect topologies, for example, left-handed rather than right-handed twist (d), or incorrect connectivity (f). These families may not be very different in energy or in native contacts, but the pattern of residues will destabilize the incorrect folds relative to the native-like fold. Only molecules with the correct fold, (e), can progress down the 'folding funnel'. Molecular chaperones and other auxiliary factors may help to avoid aggregation of the states with exposed hydrophobic residues in vivo and may help effect the rearrangement process. The folding process might involve substantial unfolding of non-productive intermediates (¢) or local reorganization of productive intermediates (6"). Some individual subdomains (cylinders with shaded caps) may reach their native-like states before others. Whether or not intermediates accumulate, or whether parallel paths are seen, ultimately depends on the energetics affecting the relative stabilities and rates of formation of these species compared with the final consolidation of the native fold. The shaded areas of the helices represent hydrophobic residues.

Collapse and cooperativity in protein folding Miranker and Dobson

also useful in its capacity to provide data for theoretical modelling. In recent work, analogues of • values based on native contacts in folding and unfolding simulations of CI2 were found to compare favourably with the experimental findings from the mutagenesis studies [8*,9°°]. Another approach to probing the nature of the transition state of a reaction is to measure activation volumes through the use of pressure-jump techniques. This approach has recently been used in conjunction with fluorescence spectroscopy to study the folding of staphylococcal nuclease [10°]. Although nuclease does not follow two-state kinetics for folding under all conditions, like many proteins it appears to be two-state under the partially denaturing conditions applied in these experiments. The system volume was found to be largest in the transition state, although more closely resembling that of the native state than that of the unfolded protein. This result has been interpreted as being consistent with a structure in which the transition state excludes solvent from its interior, while lacking the specificity necessary for a tightly formed compact state [10°]. In this, as in the above studies, experimental data have been interpreted using a simplified unimolecular protein reaction coordinate. Despite the difficulties in interpretation of such data for a system as complex as a protein, experiments of this type provide important data against which different models of folding can be tested. Furthermore, they raise a wide variety of questions about the distribution of energy throughout the available vibrational modes of a molecule, and the nature of cooperative transitions during a reactive process. Such issues are crucial even in small molecule reactions [11], and must be so in proteins. For a range of other proteins including barnase, lysozyme, ribonucleasc A, cytochrome c, myoglobin and luciferase, refolding has been found to be muhiphasic. This is the second type of kinetic behaviour found experimentally and indicates the existence of populated states intermediate between fully folded and fully unfolded structures. Such behaviour offers the opportunity to explore separate facets of folding decoupled from one another. It is interesting that in the folding of minimal lattice models, a transitioa from two-state to three-state folding is observed on manipulation of a parameter governing the strength of inter-residue attraction [12°]. A similar change has been observed experimentally following a cavity-creating mutation in the hydrophobic core of ubiquitin [13°]. The distinction between two-state and multi-state folding does not, therefore, appear to be a fundamental issue, but may depend only on whether the stabilities of intermediates are sufficiently large and barriers between states sufficiently high for experimental detection (Fig. 1). However, the ever-increasing skill with which novel techniques are developed and existing techniques improved provokes questions as to what these techniques are actually monitoring. For example, although both far UV circular dichroism (CD) and hydrogen-exchange experiments probe

33

the formation of secondary structure, hydrogen-exchange protection will only be revealed when the persistence of such structure is much greater than that necessary to produce substantial ellipticity in the CD experiment. Thus, incremental development of structure may result in apparently different kinetic behaviour when monitored by different techniques. A more detailed understanding of the correspondence between molecular structure and spectroscopic properties would, therefore, undoubtedly result in a significant increase in our knowledge of the transient structures formed during folding. For larger proteins, multiplicity of states may also represent the breaking down of the folding process into localized regions of the structure. In muhimeric systems such as luciferase [14] it is clear that folding units are well defined subunits of the protein. For single subunit proteins, however, the existence of intermediates suggests the presence and assembly of autonomous folding domains within the otherwise cooperative structure (Fig. le,f). Thus, whilst the 86-residue all-et protein ACBP folds in a single step [3°], the 153-residue all-et protein apomyoglobin has multiple intermediates corresponding to sequential folding of distinct regions of the native structure [15]. Similarly, the 67-residue all-13 protein CspB [4"] folds cooperatively, whereas such structure in the 153-residue interleukin 113 forms in several steps over a wide range of timescales [16]. In the case of large proteins, complete structure seems to be formed through the incremental assembly of domains. Further experiments are needed to establish the particular role of structural domains in the assembly of such systems, but the existence of local cooperativity prior to global cooperativity in the folding of even modestly sized proteins appears well established. There is considerable debate, however, as to the significance of intermediates, particularly as to whether they act to direct the process of folding or are off-pathway species that represent kinetic traps. Since intermediate states are not fully native, all intermediates are in one sense misfolded proteins and need to be classified on other grounds. A major criterion for classification pertains to whether an intermediate is productive or not. A non-productive intermediate is one which must adopt a previously sampled state in order to fold (Fig. lb). This is particularly difficult to characterize. A simple reaction such as U~--~I--+N (where U is the unfolded state, I is the intermediate and N is the native state), involving a productive intermediate, will provide biphasic kinetics which are indistinguishable from I+-+U---+N, a reaction involving a non-productive intermediate, if the rates establishing the equilibrium between I and U are fast compared to the rate of formation of the native state. Detection of such an equilibrium can, however, in principle be achieved using pulse labelling hydrogen-exchange experiments [17] on the assumption that amides in I and N are substantially protected from exchange. In the

34

Foldingand binding

case of hen lysozyme, no such equilibrium was found, allowing the observed biphasic kinetics to be attributed to parallel pathways [18°]. For apomyoglobin, however, the early establishment of an equilibrium between two-states was indicated by a sigmoidal transition in the magnitude of the far UV CD dead time event as a function of the denaturant in the refolding media [15]. Such experiments do not, however, distinguish between productive and non-productive intermediates and further experiments are needed to address this issue.

A key issue associated with the experimental detection of intermediates is one of timescale. T h e rate at which a particular intermediate converts to the native state can vary enormously. At one extreme, conversion is so fast as to render the intermediate undetectable. At the opposite extreme, the rate is so slow that equilibrium is never achieved. T h e latter, resulting in kinetic trapping (Fig. ld), has been implicated in the refolding of an ct-lytic protease that lacks its pro sequence [19], the homodimeric versus heterodimeric assembly of luciferase [14] and the formation of a stable monomeric as well as an intercalated dimeric form of the CD2 N-terminal domain [20"]. Thermodynamic stability is clearly an insufficient prerequisite for attainment of the native state in finite time under non-equilibrium conditions [21]. One might expect, however, that proteins evolve in such a way that there is a minimum of accessible traps along the folding pathway and that kinetic trapping, therefore, is comparatively rare. This points to the importance of characterizing the conditions and/or structural factors which give rise to kinetically trapped states [22,23°]. In the active luciferase heterodimer, for example, the recent crystal structure [24 °] shows close structural similarity between the et and 13 domains and is consistent with the specific association observed for the trapped 1~13 homodimer.

As well as the issue of multiplicity of states, the question of multiple or single pathways for folding has arisen. Two extreme possibilities exist. In one of these, multiple pathways exist and converge only at the fully native state [25]. At the other end of the spectrum is a situation in which all molecules follow the same sequence of steps in order to achieve the fully folded state. These two views affect the interpretation of experimental results. Indeed, given the heterogeneous nature of unfolded states, the definition of a single pathway needs to be relaxed for the earliest steps in folding. But in the case of lysozyme, evidence for several distinct pathways has been reported [18",26]. Here it appears that the different pathways arise because of heterogeneity in the species very early in folding, perhaps as barriers between the reorganization of alternatively folded species. An important observation in this respect is that it is not the nature of initial denatured states (Fig. la) that is important for folding, but rather the refolding conditions (Fig. lb) [3°,27°1.

D e v e l o p m e n t of structure In the case of those proteins where intermediate states exist, an important experimental objective is to characterize them in as much detail as possible. T h e apparent heterogeneity and short lived nature of transient species makes them particularly difficult to study by structural techniques. During the past year, however, very important progress has been made towards realizing this objective. Traditionally, kinetic measurements are made by rapidly mixing, on a millisecond timescale, a protein from a denaturing environment to a native one, or vice versa. T h e results of such mixes can be analyzed either directly by stopped and continuous flow optical measurements or indirectly using quenched flow techniques, notably pulsed hydrogen-deuterium exchange.

One difficulty, which arises particularly when probing early stages of protein folding, is the analysis of dead time events which are often deduced by normalization with the spectral properties of the protein under conditions not found in the refolding experiment. In practice, the unfolded protein under denaturing conditions may not have identical spectral properties as the unfolded protein under native conditions (Fig. la,b). In addition, an accurate determination of the real mixing time is extremely difficult. This can give rise to systematic errors in the time axis and, in situations where the observed kinetics are of the same order of magnitude as the mixing time, a dead time event may be artifactually found or an error may be made in determining the relative magnitude of two events in a biphasic observable. Careful work on a number of proteins including lysozyme [18 °] and cytochrome c [28] has, however, shown that the earliest states have non-rigid tertiary structure as measured by near UV CD, yet considerable secondary structure as measured by far UV CD, offering little protection from solvent exchange. Considerable effort is now being devoted to obtaining more detailed information about specific structural aspects of these intermediates using both established and novel real-time structural methods.

T h e environmentally sensitive fluorescent probe 1-anilinonaphthalenesulfonic acid (ANS) is the most widely used probe for the characterization of a solvent-exposed hydrophobic core in polypeptides. T h e presence of dead-time changes to intrinsic fluorescence intensity and the binding of ANS to a number of proteins indicates a degree of solvent exclusion characteristic of the formation of at least a rudimentary hydrophobic core. Interpretation of ANS studies is, however, complicated by the uncertain nature of the binding requirements and the possibility of perturbation of the folding pathway by the hydrophobic nature of the probe molecule. By binding to a non-native state, the stability of such an intermediate may be increased artificially by the presence of ANS. Indeed,

Collapse and cooperativity in protein folding Miranker and Dobson

recent work on carbonic anhydrase and ct-lactalbumin indicates dramatic differences between experiments in which ANS is always present during refolding and experiments in which ANS is pulsed at specific time points [29"]. For these systems, a compact hydrophobic core is sampled on the refolding pathway at the earliest stages of refolding, and the ability of kinetic intermediates to bind ANS is lost on a timescale less than a tenth that of the acquisition of the native state. A critical question concerning early species on folding pathways is the level of compactness that develops as these states fold. Fluorescence measurements have yielded a wealth of information as a result of changes to the environment of tryptophan residues or the binding of fluorescent ligands including ANS and tagged substrates [30]. At this level, fluorescence serves principally as an order parameter of the transition from unfolded to folded states. On the folding timescale, however, these experiments represent equilibrium measurements of the properties of the probe. By using pulsed lasers, the decay of fluorescence anisotropy, which occurs on a nanosecond timescale, can be measured and interpreted in terms of the dynamics of the protein populations at millisecond time points along the folding pathway [31°]. Application of this technique to monitor ANS binding to dihydrofolate reductase (DHFR) has yielded fluorescence anisotropy decay curves after 20 ms of refolding equivalent to those measured for the native protein. This indicates that the molecule is of a similar size to the native protein at this time point, although measurements by conventional optical methods have indicated that only a small fraction of native protein is present. A more direct measurement of protein compactness can be obtained from small angle solution X-ray scattering [32°]. This method allows several structural parameters to be measured, notably the multimcric state of a system and the radius of gyration. By coupling this technique with rapid mixing, assessment of the compactness of early intermediates has recently been possible in real time [33*]. In previous work on apomyoglobin, it was shown that a burst phase intermediate with substantial secondary structure as measured by CD and hydrogen-exchange protection precedes the -1 s time constant required for the protein to reach its native state [15]. Measurement of the scattering intensity spectrum after lOOms of folding provides the important finding that the protein is indistinguishable in terms of its compactness from the native state, and is clearly different from the unfolded state [33*]. Although this approach suffers from limited signal-to-noise ratios due to short data acquisition times, its further application promises to be a key part of characterizing folding intermediates. A second crucial piece of information concerning transient compact states is the extent to which stable and specific tertiary interactions are formed. In the time-resolved

35

fluorescence study of D H F R described above, anisotropy decay measurements of the five tryptophan residues collectively indicate that side-chain mobility after 20ms of refolding is still comparable to that of the unfolded state [31"]. The need for increasing detail about side-chain packing at the molecular level has recently been addressed by NMR. For systems that fold relatively slowly, NMR methods, at least in one dimension, can be used to characterize the structural transitions in real time [34,35°°]. T h e NMR spectrum of the burst phase intermediate of apo ct-lactalbumin was found, using this approach, to lack not only the chemical shift dispersion characteristic of the native state, but also the narrowness of peaks characteristic of highly unfolded proteins. Indeed, the burst-phase intermediate closely resembles the acid-induced equilibrium intermediate of the same protein, which has been shown to be a compact molten globule [35°°]. In related work, the unfolding transition of ribonuclease A was studied using N M R methods [36°']. It is reported that the protein, which otherwise shows a two-state transition as indicated by near and far UV CD, loses chemical shift dispersion within the dead time of the experiment, and then attains the unfolded state on a timescale consistent with CD. On this evidence, it has been proposed that the species formed in the penultimate step of the refolding of this protein resembles a molten globule whose core is inaccessible to water [36*°]. This fits with the idea that achieving a compact native fold is not sufficient for attainment of the native state. Furthermore, it is an important result because it challenges the idea of two-state cooperativity generally assumed to be a characteristic of the unfolding of small proteins. T h e extent to which structure formation may be described as cooperative is a vital issue in folding. Further evidence concerning cooperativity in the formation of individual elements of structure can be obtained from N M R measurements of hydrogen-exchange phenomena by comparison of the similarity in protection factors and/or kinetics within different regions of the protein. Our ability to probe this issue more directly has, however, recently been greatly enhanced by developments in mass spectrometry [37"]. Most spectroscopic methods, including NMR, yield observables that represent the average of the properties of populations of refolding proteins which are often heterogeneous. Mass spectrometry differs in this respect as it permits the resolution of populations of protein molecules whose mass has changed as a result of hydrogen-deuterium exchange. Furthermore, the distribution of masses provides a measure of cooperativity. Pulsed hydrogen exchange, for example, of a protein undergoing a cooperative transition from a state in which all labile sites are wholly unprotected from exchange to one where they are wholly protected will yield mass spectra whose peaks are shifted in mass but remain as narrow as the fully protonated protein. Peaks which are significantly broadened as a result of deuterium incorporation are the result of states that

36

Foldingand binding

are formed less cooperatively [37*]. Thus, delineation between alternative folding pathways [26] and structural characterization of the rate-limiting step [38 °] have been presented for hen lysozyme; two-state folding has been demonstrated for ACBP [3°]; and distinction of folded and misfolded conformations proved possible in a synthetic fibronectin module [39*]. Other promising advances in this methodology include its application to study the simultaneous folding of mixtures of proteins which differ in mass. In situations where the refolding properties of two proteins are marginally affected by mutation, this allows confident comparisons to be made since the experimental conditions are necessarily identical. In a study comparing avian lysozymes, for example, subtle differences in the cooperative formation of intermediates were characterized by this method and interpreted in light of the tertiary locations of the sequence variations [40°]. The concept of domains in folding, that is, regions of structure that are cooperatively formed independently of other regions, is an important one in that it raises the possibility that this is a key feature of the assembly of large proteins. In the kinetic refolding of hen lysozyme, for example, hydrogen-exchange labelling indicates that the two structural regions are formed independently of each other, at least as far as the overall fold is concerned; only at the last stage do the structural regions come together to form the fully native state [41]. Protein engineering methods for barnase have shown that the formation of a distinct region of the native structure results in the major intermediate in the refolding process and indeed is present in the transition state [42]. In equilibrium studies of cytochrome c under strongly native conditions, the kinetics of hydrogen exchange were measured as a function of denaturant at concentrations far below those needed to unfold the protein [43°°]. For this system, it is suggested that the amides can be divided into four groups corresponding to the substructures observed in the native state. This study highlights the importance of the domain nature of protein folding since in the unfolding of a system widely held to be dominated by a two-state transition, intermediates are nevertheless sampled and characterizable as subdomains of the native protein structure [43"].

Specific interactions within i n t e r m e d i a t e s The kinetic experiments described above provide clear evidence that those intermediates that accumulate during folding have developed some of the characteristics of native proteins: secondary structure, compactness and at least local cooperativity. Important issues are whether these characteristics are coupled with specific (if disordered) tertiary contacts and whether they possess an overall fold or topology close to that of the native state. If so, this may be a key element in creating the driving force that drives proteins to their native states. This topic has been addressed in a number of laboratories by studying stable analogues of transient intermediates.

The most experimentally important compact non-native protein state is the equilibrium molten globule [44°]. The molten globule has substantial secondary structure but lacks the persistent tertiary contacts characteristic of native states. It has an exposed hydrophobic core as measured by ANS binding and is nearly as compact as the native state. While this is the classical description of the molten globule, the term has recently (and somewhat controversially) been extended to include a wider range of compact states. An example of one extreme is the 'molten coil' state of a disulphide-free variant of basic pancreatic trypsin inhibitor (BPTI) in which no secondary structure is detected [45*]. Another extreme is the 'highly ordered molten globule' of interleukin 4, in which substantial persistent tertiary structure is present [46]. Therefore, there is likely to be a continuum of levels of structure possible for equilibrium intermediate states of different proteins. One approach to examining the significance of these states in folding has been to compare their properties with the characteristics of transient intermediates in non-equilibrium experiments. In this way, direct evidence for the similarity of kinetic and equilibrium molten globules has come from hydrogen-exchange protection experiments on myoglobin [15]. The real-time NMR experiments described above, recently carried out for the folding of ct-lactalbumin, add further weight to such a conclusion by showing that the chemical-shift dispersion and dynamics of a transient intermediate closely resemble those of its equilibrium molten globule [35°*]. All of this work suggests then that, at least in the case of some proteins, the equilibrium molten globule is indeed a good model of transient species formed during kinetic refolding. It is therefore of considerable importance to characterize these states in as much detail as possible.

As in transient intermediates, a key characteristic of the equilibrium molten globule is its substantially reduced size relative to the highly unfolded state. Compactness in stable states is traditionally measured using gel filtration. However, measurements have also been made using ultrasonic techniques; such a study has indicated a 40% increase in the volume of the cytochrome c A-state relative to the native state [47"]. Direct measurements of molecular size have also been performed using small angle X-ray scattering techniques demonstrating, for example, a 30% increase in the radius of the apomyoglobin molten globule relative to the native state [32"]. This value is somewhat larger than that observed in kinetic experiments on the same system [33"1 (see above), but with the present experimental error in the measurements, the significance of this is not yet clear. While much more work is needed in this area, particularly to deal with the issues of asymmetry and heterogeneity in the nature of the structure within a given state, it appears that the molten globule state is increasingly being observed to be somewhat larger than the native state, although it remains significantly smaller than the unfolded state. As such, it represents a possible

Collapse and cooperativity in protein folding Miranker and Dobson

means by which conformational space is progressively restricted in the kinetic refolding of proteins. The classical molten globule as a universal intermediate in protein folding is commonly thought of as having little or no stable tertiary structure. This conclusion is, however, now being questioned. It seems likely that a polypeptide chain with native-like secondary structure collapsed into a volume comparable to that of the native state will frequently be found to have an overall fold related to that of the native protein. If loop lengths of an up-down-up-down four-helix bundle are short, for exampie, collapse to a structure resembling up-down-down-up topology might be highly improbable. In accord with this, compact non-native states are increasingly being shown to have native-like character in their overall topology (Fig. le). A very important finding in this respect is the native-like propensity in an isolated a-domain construct of the equilibrium molten globule of ct-lactalbumin [48]. This work showed that under conditions in which random rearrangements of alternative disulphide connectivities could be made, the native disulphides are nonetheless highly favoured. Furthermore, the intact protein shows a native-like propensity to form disulphide bonds in the ct domain but not the [3 domain [49"*1, consistent with structural conclusions from hydrogen-deuterium exchange measurements [50]. Another consequence of this work is that it supports the supposition that intrinsic structural preferences, rather than disulphide connectivities, define the overall fold of a native protein. Other studies have also provided evidence for specific interactions within compact states. Analysis of a one-disulphide variant of BPTI, which contains three disulphides, shows a minimum of two interconverting conformations which have a common native-like core [51"]. In a mutagenesis study of cytochrome c, substitutions affecting the interface between two helices found in the native protein have identical effects on the free energy of transition from the native to the denatured states as from the A state to the denatured state [52°1. This has been taken as an indication that the interface is present and offers identical stabilization to both the A state and the native state. Finally, protection from hydrogen exchange in the molten-globule state of equine lysozyme is located within regions of the sequence that correspond to three of the four helices in the native state [53"]. Interestingly, contact-map analysis of the side chains of these three helices in the native protein reveals that the protected residues participate in a compact cluster within the core of the protein. Taken together, this information suggests that the core present in this molten-globule state is structurally similar to the core of the native conformation. It is generally held that attainment of the native state of a protein during refolding is irrefutably measured by recovery of the biological activity. It may, however, be more common than previously supposed that transient

37

states are sufficiently native-like to have some biological activity. Recently, it has been suggested that a kinetic intermediate in the refolding of ribonuclease A is capable of binding an inhibitor of the native protein [54*]. Similarly, the heterodimeric domains of bacterial luciferase have been shown to fold independently into structures resembling molten globules [55] and which retain residual enzymatic activity [14]. In contrast, the kinetic refolding of hen lysozyme shows formation of a native-like pattern of amide protection [26] for a fraction of the protein population at a rate for which no corresponding phase is observed in the binding of its inhibitor [30]. An important element in assessing the degree of native-state formation by such methods is, however, the requirement that the on-rate of ligand binding greatly exceeds the refolding rate of the protein. This emphasizes the need for the use of a variety of techniques to monitor the folding process. Sequence determinants Given the importance of the attainment of a native-like architecture during the folding process, a major issue that arises is how the polypeptide sequence codes for this. This is the essence of folding, and it seems clear that the basic rules relating sequence to fold must be simple as well as universal. Theoretical [56°1 and experimental [57] work has indicated that the distribution of hydrophobic and hydrophilic residues along a sequence is a major determinant of the overall folding of a protein. Evidence for this comes from the analysis of native proteins; indeed, it is the basis of predictive methods such as 'threading' [58°]. Combinatorial mutagenesis methods have similarly shown that such patterning is more important than intrinsic secondary structure propensity for the formation of helical and sheet structures in amphipathic peptides [59°]. Studies in which the hydrophobic patterning of residues is largely random can yield polypeptides with very high stabilities, judged from cooperative transitions with a high melting temperature (Tin) , and that have CD spectra characteristic of native proteins [60"]. For one of these polypeptides, however, protection from amide exchange has been measured and was found to be negligible [60°]; all protonated amides exchange with deuterium in 90 min at pH 4, behaviour typical of that seen in molten-globule states of native proteins. In contrast, amides of fully native proteins can have protection factors of 108 with residual amide proton signals persisting for months [61]. The CD and hydrogen-exchange properties of these randomly generated proteins are consistent with results seen in de novo designed proteins [62°]: namely, the proteins have only achieved a compact state, not a native one. One can infer, therefore, that natural selection is tolerant of proteins which can only form molten globules. From the point of view of stability, it is advantageous for the protein core to be disordered, because the ordering of protein side chains necessary for highly specific native states sacrifices entropy. The rapid development of techniques in this area, for example, phage display

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Foldingand binding

[63*], will undoubtedly provide new insights into the relationship between sequence and folding behaviour. T h e presence of hydrophobically induced compact states early in the refolding pathways of many proteins does not mean that such states are a necessary feature of the folding process, although it is strongly indicative of such a situation. Correspondingly, the failure to observe an early collapsed state in a given system may merely indicate that the intermediate is separated by a barrier which is too small to allow significant accumulation on the millisecond timcscale of a chemical-mix experiment (Fig. 1). Methods are, however, being developed to probe structural events occurring on faster timescales; for example, laser-induced temperature jumps applied to ribonuclease A [64*] have been used to monitor unfolding events on a nanosecond timescale. Moreover, protein sequences may exist whose initial intermediates are not compact. Given the occasional presence of buried salt bridges [65 ° ] and water, one might imagine sequences with hydrophilic cores. Perhaps there are knot-like folding motifs, whose pathways require cycles of folding and unfolding to achieve a native state. However, not only have none of these been observed, but the range of folded conformations seems to be further restricted and indeed to be dominated by a small number of superfamilies [66]. In the light of the work discussed above, it seems that natural genetic variation might easily generate collapsed structures which are sufficiently stable to escape cellular proteolysis [67"]. If it proves generally true that productive non-native states can show specific, albeit weak, binding to their substrates, generations of point mutations might then produce the highly specific tertiary contacts necessary for optimal protein activity, recognition by other cellular components and effective proteolytic turnover. Nature, it seems, has not had to find a means of solving the Levinthal paradox. Rather, the problem of solving an astronomically large combinatorial problem to find a global optimum has been bypassed by capitalizing on the intrinsic property of heteropolymers in water to collapse, forming a stable hydrophobic core. T h e sequence selection then determines the specific structural elements that evolve from this collapsed fold. One important consequence of this concept is that it suggests that the sequence may direct folding not only by stabilizing the native conformation, but also by destabilizing incorrectly folded states (Fig. 1). There is evidence for such ideas from experiments probing the interaction of leucine-zipper peptides [68]. Here, dimers, trimers and tetramers can be formed alternatively by conservative mutations which affect the steric packing at the zipper interface. A second consequence of this selection mechanism is that many different sequences having different functions could evolve from the same fold [69], a fact perhaps reflected in the apparently limited total number of distinct folds occurring in the protein database [66].

Conclusions

In this review, we have sought to highlight the developments that have been made recently in delineating and explaining the folding pathways of proteins. One of the themes discussed has been the adaptation of techniques which previously had only been applied to samples at equilibrium to the real-time observation of folding events. We believe there is much scope for further extension of this strategy in the future. T h e s e techniques have already led to an increased knowledge of molecular dimensions and to suggestions that native-like folds and specific interactions are more important in the early stages of folding than was previously suspected. A second issue relates to the role of intermediates in folding and the extent to which they are either productive and direct folding, or else represent non-productive intermediates, possibly resulting in kinetic traps. There is little doubt that the formation, even fleetingly, of a collapsed state with native-like character is an important concept in understanding the nature of the folding process, but the need to reorganize species during folding (Fig. 1) is also likely to be a key aspect of the process, particularly for more complex systems. This topic is strongly related to other important aspects of folding not covered in this review, such as the role of molecular chaperones and other helper proteins in vivo (Clarke, this issue, pp 43-50), and the relationship between non-productive folding events and a variety of pathological conditions [70"]. Another focus of attention this year has been the study of unfolding events. There appears to be considerable similarity, at least in some cases, between folding and unfolding events and studies of the latter are providing invaluable insights into assessing the significance of intermediates and the nature of later stages in folding. Finally, we have highlighted the considerable effort being directed towards the identification of those aspects of protein sequences that generate productive folds, particularly with regard to the formation of collapsed states and the specific overall architecture of the folded state.

In the face of ever-increasing detail regarding the experimental behaviour of particular systems, theoretical analyses are playing an increasingly important role in helping to interpret these data and hence in identifying key features of folding. Lattice models have been prominent in this area during the last year. Using results from such models, for example, the free energy surface on which proteins fold has been described using a minimum of parameters to give details of its ruggedness, steepness and size I71"°]. Much is being learned from these models about the relative balance of entropy and enthalpy, the detailed nature of the energy surface, the role of cooperativity in the folding process [72,73 ° ] and the coupling of the development of compactness with the formation of stable structure [74,75]. An important aspect of these approaches is the ability to map corresponding states measured for real systems and computer models to a common phase diagram

C o l l a p s e and cooperativity in protein folding Miranker and Dobson

[71°•]. In an important study, simulations of a self-avoiding chain on a cubic lattice have indicated that a well defined energetically distinct native state is necessary for folding [761. This has led to an evolutionary explanation for the low thermodynamic stabilities of fast-folding proteins [77"] as well as to a proposal that formation of a nucleus of particular contacts could be rate limiting in the formation of a native state [78]. Significant in larger systems, the latter observation manifests itself as multidomain folding [79*] and correlates well with similar observations from experimental studies. A particularly interesting finding has been the apparent correlation between the experimentally observed existence of parallel refolding pathways and similar features of off-lattice models [80°]. In the latter, a fraction of the molecules form a nucleus, leading to the formation of the native state on a relatively fast timescale compared to the rest of the population, which forms structure more slowly. T h e synergetic interaction of theory and experiment represented by such examples will undoubtedly continue to be an important aspect of efforts to understand fully the complex problem of how a protein folds.

Acknowledgements AI)M is a Junior Research Fellow of('hrist Church, Oxford. The research of C M I ) is supported in part by an International Rcscarch Scholars award from thc Howard Hughcs Medical lnstitutc. T h e Oxford Ccntrc for Molecular Sciences is supportcd by the UK Biotcchnology and Biological Scicnccs Research Council, the Medical Rcscarch Council and the Enginccring and Physical Scicnccs Rescarch Council.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • ••

of special interest of outstanding interest

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Jackson SE, Elmasry N, Fersht AR: Structure of the hydrophobic core in the transition state for folding of chymotrypsin inhibitor 2: a critical test of the protein engineering method of analysis. Biochemistry 1993, 32:11270-11278.

3. •

Kragelund BB, Robinson CV, Knudsen J, Dobson CM, Poutsen FM: Folding of a four-helix bundle: studies of acyl-coenzyme A binding protein. Biochemistry 1995, 34:7217-7224. The refolding of a four-helix bundle is characterized by several stopped and quenched flow techniques as rapid (<5 ms at 25°C) and two-state. Refolding from guanidine hydrochloride and acid-denatured states was found to be closely similar. This behaviour was associated with the absence of slow reorganization steps in the folding process of this small protein, 4. •

the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding. Proc Nat/Acad Sci USA 1994, 91:10422-10425. The transition state of the two-state refolding of CI2 is characterized by analyzing the kinetic and equilibrium effects of 74 mutations at 37 sites. Most mutations result in a significant decrease in the localized stability of the transition state compared to the folded protein, The transition state is interpreted as resembling an expanded form of the native protein structure. "7.

Li AJ, Daggett V: Characterization of the transition state of protein unfolding by use of molecular dynamics: chymotrypsin inhibitor 2. Proc Nat/Acad Sci USA 1994, 91:10430-10434. Molecular dynamics simulations of CI2 in water were performed at elevated temperatures to characterize the unfolding of this protein. An analogue of experimentally determined ~ values was developed and corresponded well with the experimental findings. A structural model of the transition state of this protein is put forward. 9. •-

Itzhaki LS, Otzen DE, Fersht AR: The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleation-condensation mechanism for protein folding. J Mol Biol 1995, 254:260-288. This study extends earlier work on CI2 and notably draws attention to a possible nucleation site predicted independently in lattice model simulations. 10. •

Vidugiris GJA, Markley JL, Royer CA: Evidence for a molten globule-like transition state in protein folding from determination of activation volumes. Biochemistry 1995, 34:4909-4912, The folding and unfolding kinetics of nuclease A have been measured afler jumps in pressure. The relationship between kinetics and pressure permit the relative activation volumes of native, unfolded and transition states to be determined. The results are taken to indicate a transition state which is enlarged compared to the native state, is loosely packed and excludes solvent. 11.

Gutin AM, Abkevich Vl, Shakhnovich El: Is burst hydrophobic collapse necessary for protein folding? Biochemistry 1995, 34:3066-3076. Two sets of lattice-model simulations of protein folding were performed that differed in the magnitude of attraction between the residues. In the case of strong attraction between residues, formation of the native conformation is preceded by collapse. For weak attraction, two-state transitions dominate. 13. •

Khorasanizadeh S, Peters ID, Roder H: Effect of amino acid changes at the helix-sheet interface of ubiquitin on the thermodynamics and kinetics of folding. Protein £ng 1995, 8(suppl):21. Meeting abstract describing the effects on refolding of ubiquitin that result from mutations to residue 26 in the core of this protein. Highly destabilizing cavity-forming mutants (Val--~Ala, Val--)Gly) are reported to change the refolding kinetics from three-state to two-state. 14.

Sinclair JF, Ziegler MM, Baldwin TO: Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1994, 1:320-326.

15.

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Baldwin RL: Pulsed H/D-exchange studies of folding intermediates. Curt Opin Struct Bio11993, 3:84-91.

5. •

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Fersht AR, Itzhaki LS, Elmasry N, Matthews JM, Otzen DE: Single versus parallel pathways of protein-folding and fractional formation of structure in the transition-state. Proc Nat/Acad Sci USA 1994, 91:10426-10429.

8. •

Schindler T, Herder M, Marahiel MA, Schmid FX: Extremely rapid protein folding in the absence of intermediates. Nature Struct Bio11995, 2:663-673. Equilibrium and kinetic studies over a wide range of denaturant conditions are consistent with two-state refolding behaviour of a five-stranded ~ barrel. Under optimal conditions, the folding is fast (<1.5 ms). Fersht AR: Characterizing transition states in protein folding: an essential step in the puzzle. Curr Opin Struct Bio11995, 5:79-84. A review of the application of site-directed mutagenesis techniques to the exploration of protein folding. Particular attention is paid to the characterization of the transition state in barnase and CI2.

39

Radford SE, Dobson CM: Insights into protein folding using physical techniques: studies of lysozyme and c¢-Iactalbumin. Philos Trans R Soc Lond Bio/1995, 348:17-25. Review including a schematic folding pathway for hen lysozyme on the basis of data from a wide variety of experimental techniques.

20. ••

Baker D, Sohl JL, Agard DA: A protein folding reaction under kinetic control. Nature 1992, 356:263-265.

Murray A J, Lewis S J, Barclay AN, Brady RL: One sequence, two folds: a metastable structure of CD2. Proc Nat/Acad Sci USA 1995, 92:7337-7341. The N-terminal domain of the lymphocyte cell-adhesion molecule CD2 was expressed recombinantly as part of a glutathione S-transferase fusion pro-

40

Folding and binding

tein. A dimeric form of CD2, found as 15% of the expressed protein, was isolated and crystallized. Investigation of the structure revealed a dimer whose domains strongly resemble the native protein, although each domain contains chains from both monomers. 21.

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22.

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23. An

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25.

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28.

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29. ,,

30.

Itzhaki LS, Evans PA, Dobson CM, Radford SE: Tertiary interactions In the folding pathway of hen lysozyme: kinetic studies using fluorescent probes. Biochemistry 1994, 33:5212-5220.

Jones BE, Beechem JM, Matthews CR: Local and global dynamics during the folding of Escherichia coil dihydrofolate reductase by time-resolved fluorescence spectroscopy. Biochemistry 1995, 34:186"7-18"7"7. Time-resolved fluorescence spectroscopy is used to measure anisotropy and fluorescence lifetimes of tryptophan residues and the bound hydrophobic reporter group ANS during the refolding of DHFR. Results are interpreted in terms of both localized and global changes to the size and dynamics of the protein during refolding. 31. •

32. ,,

Kataoka M, Nishii I, Fujisawa T, Ueki T, Tokunaga F, Goto Y: Structural characterization of the molten globule and native states of apomyoglobin by solution X-ray scattering. J Mo/Biol 1995, 249:215-228. A detail solution X-ray scattering study of holo and apo forms of myoglobin. A bimodal distance distribution is also reported for the trichloroacetate-stabilized molten globule state of the apo protein and is interpreted as reflecting loose structure about a stable core.

33. •

Eliezer D, Jennings PA, Wright PE, Doniach S, Hodgson KO, TsurutaH: The radius of gyration of an apomyoglobin folding intermediate. Science 1995, 270:487-488. The compactness of an early intermediate in the refolding of apomyoblogin is measured directly using time-resolved X-ray scattering and found to be similar to that of the native state. 34.

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35. •.

Balbach J, Forge V, Van Nuland N/U, Winder SL, Hore PJ, Dobson CM: Following protein folding in real time using NMR spectroscopy. Nature Struct Bio11995, 2:865-8?0. The refolding of apo a-lactalbumin is monitored in real time by NMR. The spectrum of the burst-phase intermediate closely resembles the spectrum of the equilibrium molten globule of this protein. Furthermore, all time-resolved spectra can be reconstructed from linear combinations of native and burstphase spectra, suggesting a cooperative transition from the burst state to the native state with no evidence for intermediates (e.g. domains) having fully native-like packing. 36. •.

Kiefhaber T, Labhardt AM, Baldwin RL: Direct NMR evidence for an intermediate preceding the rate limiting step in the unfolding of ribonuclease A. Nature 1995, 375:513-515. Using rapid-mix techniques coupled with NMR, it is reported that in the unfolding of ribonuclease A, chemical-shift dispersion is lost within the dead time of the experiment followed by attainment of the unfolded state on a timescale consistent with monophasic unfolding measured by CD. This is reported as consistent with the unfolding process, having an initial intermediate which excludes solvent and is largely native-like, but has expanded enough to allow the motions necessary to remove chemical-shift dispersion. 3"7. •

Miranker A, Robinson CV, Radford SE, Dobson CM: Investigation of protein folding by mass spectrometry. FASEB J 1996, in press. A review of the recent and rapid developments in mass spectrometry which are relevant to the study of protein folding. 38. •

Eyles S J, Radford SE, Robinson CV, Dobson CM: Kinetic consequences of the removal of a disulfide bridge on the folding of hen lysozyme. Biochemistry 1994, 33:13038-13048. Removal of the terminal 6-1 2"7 disulphide bond resulted in a protein with a similar refolding rate to the native protein. However, the compact intermediate attributed to the largely c(-helical domain seen in the refolding of the native species was completely absent. This suggested that formation of stable B-sheet structure is rate limiting for this protein. 39. •

Muir TW, Williams M J, Kent SBH: Detection of synthetic protein isomers and conformers by electrospray mass spectrometry. Anal Biochem 1995, 224:100-109. in the total chemical synthesis of the tenth type III module from fibronectin, a distinction between folded and misfolded products is made from both the charge distribution and the hydrogen-exchange properties detected by mass analysis. 40. •

Hooke SD, Eyles S J, Miranker A, Radford SE, Robinson CV, Dobson CM: Cooperative elements in protein folding monitored by electrospray ionization mass spectrometry. J Am Chem Soc 1995, 117:"7548-"7549. The ability of mass spectrometry to separate on the basis of mass is used to compare the relative refolding properties of closely related lysozymes to a high level of accuracy. This approach guarantees that experiment and analysis conditions are identical. 41.

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45. •

Ferrer M, Barany G, Woodward C: Partially folded, molten globule and molten coil states of bovine pancreatic trypsin inhibitor. Nature Struct Biol 1995, 2:211-217. A one-disulphide and a no-disulphide analogue of BPTI are characterized. The one-disulphide intermediate is denatured at pH 2.5 and forms an acid state with molten globule characteristics at pH 1.5. The no-disulphide analogue has no significant secondary or tertiary structure as measured by CD, yet is able to bind ANS, suggesting the presence of hydrophobic surface. 46.

Redfield C, Smith RAG, Dobson CM: Structural characterization of a highly ordered molten globule at low pH. Nature Struct Biol 1994, 1:23-29.

4"7. •

Chalikian TV, Gindikin VS, Breslauer KJ: Volumetric characterizations of the native, molten globule and unfolded states of cytochrome c at acidic pH. J Mol Big/1995, 250:291-306.

C o l l a p s e and cooperativity in protein folding Miranker and Dobson

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Peng ZY, Kim PS: A protein dissection study of a molten globule. Biochemistry 1994, 33:2136-2141.

49. •=

Wu LC, Peng ZY, Kim PS: Bipartite structure of the (~-Iactalbumin molten globule. Nature Struct Biol 1995, 2:281-286. Two site-directed mutants of c(-lactalbumin are designed such that two of the four disulphides are retained in either one or the other of the two subdomains of this protein. Both mutants demonstrate molten-globule characteristics in common with the native protein under acid conditions. Disulphide rearrangement studies indicate native-like disulphide formation in only one of the subdomains. 50.

Chyan CL, Wormald C, Dobson CM, Evans PA, Baum J: Structure and stability of the molten globule state of guinea-pig c¢lactalbumin: a hydrogen exchange study. Biochemistry 1993, 32:5681-5691.

51. •

Barbar E, Barany G, Woodward C: Dynamic structure of a highly ordered B-sheet molten globule: multiple conformations with a stable core. Biochemistry 1995, 34:11423-11434. NMR analysis of the molten-globule state of a one-disulphide derivative of BPTI reveals two exchange peaks for many residues. Residues representing a two-stranded 13sheet in the native protein do not give exchange peaks. This is taken as an indication of multiple conformations which share a common structural core. 52.

Marmorino JL, Pielak G J: A native tertiary interaction stabilizes the A state of cytochrome c. Biochemistry 1995, 34:3140-3143. he thermodynamic properties of unfolding cytochrome c from its native state are compared to those of unfolding from its acid denatured state. Mutations directed at the N- and C-terminal helical interface yield similar perturbations to the two transitions. This is interpreted as showing that this helix-helix interaction equivalently stabilizes the native and acid states of this protein. 53. •

Morozova LA, Haynie DT, Arico-Muendel C, Dael HV, Dobson CM: Structural basis of the stability of a lysozyme molten globule. Nature Struct Biol 1995, 2:871-875. The pattern of hydrogen-exchange protection of the acid-induced molten globule of equine lysozyme is compared to that of the native protein. The correlation of the most highly protected residues of this molten globule with the compact core of the native protein is taken as an indication of significant native-like interactions within the hydrophobic core. 54. •

Udgaonkar JB, Baldwin RL: Nature of the early folding intermediate of ribonuclease A. Biochemistry 1995, 34:4088-4096. Inhibitor binding and hydrogen-exchange protection are reported as consistent with the presence of well developed tertiary structure in an early intermediate of ribonuclease A. 55.

Flynn GC, Beckers CJM, Baase WA, Dahlquist FVV: Individual subunits of bacterial luciferase are molten globules and interact with molecular chaperones. Proc Nat/Acad Sci USA 1993, 90:10826-10830.

56. •

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Kamtekar S, Schiffer JM, Xiong HY, Babik JM, Hecht MH: Protein design by binary patterning of polar and nonpolar amino acids. Science 1993, 262:1680-1685.

58. •

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41

suggest that periodicity is a stronger determinant of secondary structure than is intrinsic propensity. 60. •

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62. •

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67. •

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Folding and binding

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