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Experimental investigation of sidechain interactions in early folding intermediates
Review
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Experimental investigation of sidechain interactions in early folding intermediates Markus Engelhard and Philip A Evans Kinetic studies of folding sometimes reveal very rapid spectroscopic changes that may indicate the population of intermediates, but it is difficult to elucidate in detail the nature of the interactions involved. In this review, we focus on one important aspect of this problem: how to probe the nature and extent of clustering of hydrophobic sidechains. As the information obtainable from different experimental approaches is outlined, it becomes clear that a combination of methods is likely to be necessary to build up a reasonable picture of early folding events. Address: Department of Biochemistry and Cambridge Centre for Molecular Recognition, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK. E-mail addresses: Markus Engelhard, [email protected]; Philip A Evans, [email protected] Electronic identifier: 1359-0278-001-R0031 Folding & Design 01 Apr 1996, 1:R31–R37 © Current Biology Ltd ISSN 1359-0278
Introduction
Protein folding involves both ordering of the backbone into specific, often hydrogen-bonded, structural elements and packing together of sidechains, especially non-polar groups driven to cluster by the hydrophobic effect. Although these are highly interdependent in native protein structures, generalized mechanistic models tend to emphasize the role of one or other aspect in initiating productive folding. The framework model, for example, suggests that elements of locally ordered backbone structure develop initially and then associate to form a tertiary structure [1]. In contrast, the hydrophobic collapse model suggests an overall condensation of the chain driven by clustering of non-polar groups and organization of the backbone within this collapsed state to form the native structure [2]. There is a clear need for detailed experimental evidence to resolve the issue of what really happens in the early stages of folding. Intermediates have been detected experimentally in the folding of many proteins and methods including protein engineering and pulsed hydrogen exchange labelling have been developed for their detailed characterization [3,4]. In the main, intermediates that have been mapped out in detail have turned out to be rather highly organized, with substantial native-like structure, and probably represent relatively late stages in folding. Earlier intermediates, which might tell us more about how folding begins,
remain more enigmatic. Some events are too fast to be studied with current methodologies, but relatively early intermediates, formed within the dead time of a stoppedflow experiment, for example, are often detectable, and understanding their nature would be an important step forward. The problem is to find experiments to characterize species that may be structurally highly heterogeneous and far from native-like. Probes of backbone folding are quite well established. Circular dichroism (CD) can provide reasonable estimates of secondary structure content and can be applied in stopped-flow studies, though it cannot tell us which residues are actually involved [5,6]. More detailed mapping of secondary structures may be possible through hydrogen exchange labelling or protein engineering, although structural heterogeneity and limited stability are problematic for both methods. The problem we are concerned with in this review is the complementary one of how to investigate the nature and extent of sidechain interactions in early intermediates. A wide variety of spectroscopic, and other, probes have been used to follow folding reactions and most are potentially sensitive to changes in the environment of one or more sidechains. Thus, detection of any change at a rate faster than overall folding is commonly interpreted in terms of population of a folding intermediate. However, most methods actually provide very little detail about the interactions involved. In looking at the earliest detectable events in a stopped-flow experiment, it may be difficult to distinguish between simple changes in the properties of a probe as its solvent environment is changed, which might be of no consequence for the folding mechanism, and potentially more significant events such as local or global hydrophobic collapse. Actually mapping the sidechain clusters is a much more difficult problem, but one that must be addressed if we are to establish not just when condensation happens but how important an aspect of folding it really is. For example, a non-specific ‘stickiness’ of hydrophobic groups might be quite incidental or even a positive obstacle to be overcome in folding. Global collapse, on the other hand, might help to ensure efficient folding by favouring an approximation of the native chain topology at an early stage in the pathway. We need detailed structural information to gain a better perspective on these possibilities. Our focus in this review is therefore on the information content of the various methods available for looking at sidechain interactions in the early stages of folding.
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Probe accessibility methods
The presumed driving force for sidechain clustering, diminished interaction with water, also provides one strategy for its detection: if one participating group is a suitable probe, such as a fluorophore, it may be possible to detect the change in its solvent accessibility. Tryptophan and tyrosine residues are intrinsic fluorophores and their emission intensities, which are sensitive to interactions both with solvent and with other sidechains, often change markedly during folding. Intensity changes are most often used to monitor formation of the native state, but additional kinetic phases associated with faster processes are sometimes apparent. Changes occurring within the dead time of a stopped-flow experiment can also be measured, although this depends on careful extrapolation of the fluorescence of the starting denatured state to the conditions of refolding [7,8]. The sensitivity of fluorescence intensity to a variety of environmental factors means that it should never be interpreted in specific structural terms [9], but other fluorescence parameters may provide a more straightforward reflection of solvent accessibility. The wavelength of maximal emission, lmax, of tryptophan (but not tyrosine) residues is influenced by polar interactions in the excited state which are effective in water but typically much less so in the hydrophobic protein interior. Hydrophobic contacts formed by the indole ring can thus be detected by a blue shift in its emission spectrum [9]. This has not yet been widely exploited in kinetic studies because most stopped-flow fluorescence set-ups simply integrate total emission, rather than generating spectra. With the increased availability of diode array detection to provide wavelength-dependent data, it seems likely that the method will be explored more widely in the future.
tions cannot usually be resolved and assigned. A more global picture of the distribution of clusters may be obtainable by using protein engineering to introduce (and, if necessary, delete) tryptophans at different places in the structure and compare the time-dependent accessibility changes of the different probes [8,11]. These experiments depend on the kinetics and mechanism of folding not being seriously perturbed by the mutations. This is not always easy to achieve, because the fused ring system of tryptophan is seldom a very conservative substitution. Careful controls are needed, therefore, in interpreting these experiments. Other probe accessibility methods have also been used, notably chemical labelling methods. A potential advantage here is that if a reaction is effectively irreversible, analytical methods can subsequently be used to characterize the pattern of reactivity of multiple probes within a single molecule. This idea is essentially the same as that of hydrogen exchange labelling, but in that case most of the probes are backbone NHs, so that only indirect implications about sidechain interactions are obtained. Important exceptions to this are the indole NHs of tryptophan residues, protection of which has indeed been detected in some early folding intermediates [12]. Other labelling methods are based on reactions of sidechain functional groups, most notably the thiol group of cysteine, which can react very rapidly as a nucleophile. Selective mutation of different sidechains to cysteine has been used as a way of introducing this probe into proteins and differential reactivity has been observed in both kinetic and equilibrium intermediates and has been interpreted in terms of accessibility to the labelling reagent [13,14]. Probe mobility methods
Another relatively simple way to obtain a more direct measure of fluorophore accessibility uses small molecule co-solutes such as iodide or acrylamide as quenchers [9]. As quenching is a contact interaction, it will be diminished if the accessibility of a fluorophore is restricted by hydrophobic clustering, as has indeed been detected in early folding intermediates of some proteins [7,10]. However, the extent of quenching may also vary with changes in fluorescence lifetime and, if the quencher is charged, in the electrostatic environment, so this provides only a qualitative measure of accessibility changes during folding. As with any additive to the medium, it is important to design experiments to ensure that the quencher does not perturb the folding kinetics [7]. In proteins having only a single tryptophan, these methods can potentially provide specific information about the involvement of that residue in hydrophobic clusters; where there are multiple fluorophores, only more general conclusions can be drawn, as individual contribu-
Another approach to the detection of sidechain interactions depends on the reduction in mobility that clustering entails. Various spectroscopic methods are available that are sensitive to molecular motions, though not all are immediately applicable to proteins. Intrinsic fluorescence is certainly influenced by dynamics, but these effects cannot be distinguished from other factors in simple steady-state fluorescence intensity measurements. Much more detailed information is obtainable from measurements of fluorescence decay rates and depolarization, for which simple models are available that allow interpretation in terms of sidechain dynamics. This approach is experimentally much more demanding than steady-state fluorescence, but it has recently been shown to be capable of providing a detailed reflection of the gradual immobilization of tryptophan sidechains as folding progresses [15]. Other techniques that have been used to detect mobility changes during refolding have used chemical modification to introduce a suitable reporter group. In particular, elec-
Review Sidechain interactions in early folding intermediates Engelhard and Evans
tron paramagnetic resonance (EPR) can be used to monitor the mobility of spin labels [16]. The relaxation rates of the electron spin are very sensitive to dynamics and it has been shown that loss of signal, corresponding to a broadening of the spectrum, provides a good probe for immobilization. In early applications, probes were introduced by non-selective sidechain modification [16], but the method can be refined by using site-directed mutagenesis to introduce reactive residues such as cysteine at a range of sites in the sequence, allowing spin-labels to be introduced at strategic places in the structure [14]. The spin labels used in these experiments tend to be substantial hydrophobic groups themselves, so that a potential complication arises from their ability to participate directly in hydrophobic clustering interactions. The principal problem with mobility-based approaches, even more than with those dependent on accessibility changes, is that it is hard to gauge the extent to which the effects observed reflect significant folding events, as opposed to strictly local changes in conformational freedom. Introduction of probes at a range of sites goes some way to addressing this problem, but it is still difficult to know how extensive and how fixed a set of interactions is implied by a given change in the experimental parameters observed. Interaction with extrinsic hydrophobic probes
An alternative to monitoring the behaviour of probes within a folding molecule is to employ external agents capable of interacting with the protein in a conformationdependent manner. An obvious example is the use of ligands of the native state to detect the formation of a functional binding site. In most cases, this effectively measures the formation of the native state [7,17], although relatively localized binding sites, such as metal-binding loops, may become organized at an earlier stage in folding [18]. A different type of extrinsic probe that has achieved popularity in folding studies is exemplified by 8-anilino-1naphthalenesulphonate (ANS), a hydrophobic fluorescent dye [16,19]. Its fluorescence is effectively quenched in aqueous solution, but the quantum yield increases sharply on binding to a hydrophobic surface. It has been found that non-polar groups in unfolded proteins are too diffuse to allow significant interaction with ANS, whereas in fully folded proteins, unless they have hydrophobic binding regions for functional reasons, the non-polar groups are inaccessible to the dye as they are sequestered in the interior. In contrast, some intermediates can interact very effectively, causing large enhancements in ANS fluorescence, presumably because non-polar groups are now clustered but with sufficient exposure to the outside or with sufficient flexibility in the structure to allow access to the dye. This remarkable kind of specificity has led to widespread applications for the method in identifying partially folded states of proteins [7,16,19,20].
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In kinetic studies of folding, ANS fluorescence has generally been investigated simply by including the dye in the refolding buffer [20]. Time courses observed in this way for refolding of several proteins were qualitatively remarkably similar: a moderate dead-time fluorescence increase was followed by a further, slower, rise before the signal gradually returned to a low level as the protein reached its native state. These results were interpreted on the basis of two key assumptions: first, that the dye bound instantaneously, so that any time-dependent effects could be assumed simply to be reporting conformational changes in the protein; second, that interaction with the dye would significantly perturb neither the folding pathway nor its kinetics. Unfortunately, it turns out that neither assumption is always valid: detailed studies of two proteins revealed that some kinetic phases observable through ANS fluorescence enhancement are actually due to the formation or reorganization of dye–protein complexes, rather than simply reflecting the progress of folding [21]. Further, it was clear that ANS could indeed profoundly alter the folding kinetics, presumably by stabilizing intermediates to which it binds. These problems can be alleviated by using a pulse method, where the ANS is introduced only after folding has been allowed to proceed for a given time, and folding curves are reconstructed by plotting the initial fluorescence then observed, as a function of the folding time [21]. Additional information from ANS binding is obtainable by applying time-resolved fluorescence measurements which can provide insight into the mobility of the bound dye molecule [15]. Global compactness
The techniques discussed so far are relatively straightforward to apply and can provide good evidence for some kind of hydrophobic clustering. They do not, however, provide information as to the extent of clustering and cannot be used to map the residues involved. There are various ways to estimate the extent of global condensation of a protein molecule, but they are not simple spectroscopic methods and their application to kinetic intermediates has been a demanding problem. In exceptional cases where intermediates are very long-lived, it has been possible to use gel permeation chromatography to demonstrate their compact structures [22], but methods of this kind are clearly inappropriate for the ephemeral early intermediates that are commonly encountered in the folding of small proteins. Small-angle X-ray scattering is a powerful method for studying the compactness of macromolecules and has been applied to several studies of equilibrium partially folded states, but kinetic applications are in their infancy. The principal limitation is the low sensitivity of the technique, but improved radiation sources and experimental designs promise much in the future [23]. Quasielastic light scattering can be used to study translational mobility, and
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hence compactness, during folding, although again this method is in the early stages of development: good evidence for almost native-like condensation of a partially folded state of lysozyme was obtained, although a long dead time (around 1 s) meant that this corresponded to a relatively late intermediate [24]. There seems to be no fundamental barrier to extending either of these approaches to the point where following the kinetics of compaction would be a realistic possibility. Proximity determination methods
If we actually want to map the structure present in a folding intermediate, the most direct way is to determine approximate average distances between specific residues. This is, of course, the basis of the standard NMR method for solution structure determination, where NOEs can provide a wealth of distance restraints from which detailed structures can be calculated. Progress is being made in applying NMR to kinetic folding intermediates, although applications are likely to be limited to relatively long-lived species [25,26]. Other ways to derive proximity information are based on much more limited numbers of specific probes. The trapping of disulphide intermediates in the oxidative refolding of proteins is a classic example. Formation of a specific disulphide with sufficient population to allow it to be trapped requires it to be stabilized by cooperation with non-covalent interactions among sidechains around the participating cysteines, so this is effectively a direct means of detecting hydrophobic clusters [27]. Further, intermediates with subsets of disulphides can be trapped and isolated, or modelled through protein engineering, so that their conformations can be studied in detail by, for example, NMR [28]. Oxidative refolding is typically much slower than that of small globular proteins lacking disulphides, but this does not mean that the mechanisms involved are fundamentally different, only that events tend to be slowed down by the thiol chemistry involved. This approach is potentially very powerful for studying disulphide-containing proteins; it has been applied in great detail in one case, but it is to be hoped that it can be extended to other systems and that the conclusions drawn can be integrated more fully with those deriving from other techniques. Another phenomenon that can provide proximity information is fluorescence energy transfer. This idea has been used in a general way by introducing dansyl groups nonselectively and monitoring the development of their emissions when tryptophans in the protein were excited: the efficiency of energy transfer was observed to increase markedly during folding, presumably reflecting shorter average distances between the donor and acceptor groups [16]. In some cases, it is possible to generate a specific donor/acceptor pair, where unique residues are present, or
can be genetically introduced, that are suitable for modification. For example, energy transfer between a single tryptophan and a single labelled cysteine allowed detection of an early compact intermediate in the folding of an immunoglobulin light chain [29]. Measurement of individual average distances requires such unique donor/acceptor pairs and the capability to make time-resolved fluorescence measurements. This idea has been applied to a variety of equilibrium non-native states of one protein, in which different probe combinations could be used to derive distance information, allowing conclusions about the extent of condensation to be drawn [30]. Protein engineering
The principal limitation of most of the physical and chemical methods discussed above is that they can provide only limited residue-specific information. The protein engineering approach, on the other hand, offers the potential to examine quantitatively how the interactions of any chosen sidechain contribute to the stability of folding intermediates and transition states. A simple detection method such as fluorescence can be used to follow overall folding, without any need to observe the sidechain directly. Applied in a systematic way, this can provide an extraordinarily detailed map of these species [3]. The prerequisite for application of the method is a relatively simple kinetic scheme, in which it can be assumed that the native and unfolded states, sometimes a metastable intermediate, and the transition states in between are all well defined and structurally independent of the solution conditions. The idea then is that selectively mutating an individual sidechain will perturb the relative stabilities of the various states in a way that depends on the extent to which the residue concerned is involved in the folded structure in each case. These free energy perturbations can be measured from a combination of equilibrium unfolding and kinetic unfolding/refolding data. This approach has been applied intensively to a small number of systems, allowing detailed mapping of the interactions present in the transition states. In one case, barnase, the presence of a well populated intermediate was also inferred and its structure mapped out [31]. In all of these instances the structure present appeared to be native-like but ‘weakened’ to varying degrees. It is possible to check for non-native interactions through analysis of so-called double mutant cycles [32], and where this has been done the assumption of native-like structure has largely been validated. The information deriving from these studies has been uniquely detailed and the method has other advantages in that, for example, it provides direct information as to the kinetic significance of the intermediates concerned. It is clear, however, that the species so far investigated in this way have rather highly developed structure, at least
Review Sidechain interactions in early folding intermediates Engelhard and Evans
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niques suggest a series of different partially folded forms, it would be difficult to obtain comparably detailed information; at the very least, it would be necessary to use different detection methods to pick out particular intermediates.
locally, and hence represent relatively advanced stages of folding. In principle, it should be possible to extend the method to earlier intermediates, but there are a number of potential difficulties and it will be interesting to see how effectively it can be applied to intermediates that other techniques suggest are not highly native-like. The use of a single spectroscopic probe to monitor transition between unfolded and fully folded states has been an attractive aspect of the applications so far, as it allows simple and sensitive methods to be applied. The data thus obtained are sufficient only if a maximum of one populated intermediate is assumed, however. In cases where other tech-
Detailed interpretation of protein engineering experiments depends on two key assumptions: any folding interactions present should be native-like and the mutations made should be non-disruptive of the structures of the species involved. These points will both have to be examined carefully in extending the approach to early interme-
Figure 1
I
unfolded state
II
native state
= tryptophan = sidechain contributing to the principal hydrophobic core
Trp fluorescence a
= covalently linked fluorophore
Intensity changeb
?
?
?
Accessibilityc Mobility ANS bindingd
X
?
X
X
X
Compactness Energy transfere Native functional properties
A hypothetical folding pathway in which two intermediates — I, involving local clustering of groups of residues, and II, a globally collapsed, molten globule state — are sequentially populated. This is not proposed as a general model for folding, nor would the population of the intermediates necessarily be kinetically resolved, but it serves to illustrate the different aspects of folding that might be detected by some of the probes discussed in the text. aA single tryptophan fluorophore is used as an example of a ‘local’ structural probe. Other such probes responding to accessibility or mobility changes are discussed in the text. bAs fluorescence intensity depends on a variety of environmental factors, changes in each stage of folding are likely,
X
but their magnitudes and directions cannot be easily predicted. cIn this particular hypothetical protein, the Trp sidechain becomes involved in a cluster at an early stage, with consequent accessibility and mobility changes. Detection of such an intermediate, however, is partly a matter of chance — this early intermediate might escape detection by such a probe if the latter were located elsewhere in the sequence. dIt is not clear how extensively structured an intermediate needs to be to bind ANS efficiently — thus it is uncertain whether the early intermediate would be detected. eFluorescence of a covalently linked fluorophore as a result of energy transfer from the single Trp can be used to probe changes in the average separation of the two groups.
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diates. Effects of non-native interactions can be detected through double mutant cycles [32], but it is hard to pin down any particular non-native structure that may be present. This has not proved to be a serious limitation in the applications reported to date, but it remains to be seen whether this will be true in states further from the native structure. Relaxation of folded and partially folded structures in response to mutations has been observed to diminish the consequent free energy perturbations to varying degrees. Again, these effects have not been a serious barrier to application of the method so far, but if the structure of an early intermediate were very flexible, the capacity for rearrangement and compensation for mutations might be correspondingly greater. Thus, in cases where protein engineering suggests that interactions of a particular sidechain are substantially absent in an intermediate, it will be necessary to take care to eliminate the alternative possibility: that the interactions it forms are just not very specific. Conclusions
Many techniques are now available that can contribute to developing our understanding of how sidechains begin to interact in early folding intermediates. They are clearly not all sensitive to structure at the same level; for example, states that bind ANS may nonetheless have highly mobile tryptophan sidechains [15]. Thus, no single method can provide a complete picture and it is clearly desirable that data from a variety of approaches should be assembled before drawing too many firm conclusions in a particular case (see Fig. 1). If our understanding of the overall organization of folding is to benefit significantly from studying this behaviour, we will need a much more detailed picture of the specific interactions present than has hitherto been achieved for early intermediates. Experiments that can provide this information directly are very demanding and it seems unlikely that more than a few approximate distance constraints will be obtainable in any given case. The indirect evidence obtained from protein engineering can potentially provide the solution to this problem, as it has in the characterization of more highly structured species on folding pathways, although there are likely to be a number of difficulties if the set of interactions present has significant flexibility. Perhaps the key to making sense of the data we can obtain from kinetic experiments on early folding intermediates is to draw them together with results from alternative approaches. Equilibrium denatured states and protein fragments provide a very important point of comparison. Many of the same indicators of partial structuring observable in early kinetic intermediates, such as diminished probe accessibility and mobility and ANS binding, are also observed in some of these species and it seems very likely
that they share many similar properties. The big difference, however, is that these equilibrium states can be studied at leisure and are therefore much more amenable to methods such as NMR that are intrinsically slow but can potentially provide a wealth of structural detail. The significance of the other spectroscopic effects should be much clearer in the light of this kind of information. A further point is that the very earliest events in folding are much too fast to be resolved by conventional methods and it may well be the case that studying fragments, where folding is effectively halted at a particular stage for want of the other residues needed for wider structure development, will be the only way to understand what is likely to happen on this timescale. Theoretical methods for predicting folding mechanisms constitute another important complementary approach. There are two broad classes of approach. One is to analyze the native structure and devise an algorithm that will identify aspects of the structure that might be formed at an early stage of folding. These methods necessarily involve a great deal of assumption about the general nature of initiation events, so it is clear that they should be developed closely in parallel with the growth of our understanding deriving from experimental evidence. Other methods actually aim to simulate all or part of the folding pathway, using either a simplified model for the protein molecule or explicit molecular dynamics; in these cases too, comparison with experimental data will be vital to determine the extent to which the models set up are a useful approximation. This field has seen a great deal of activity and it seems certain that there will be major developments in our understanding of the early events in folding in the coming years. It remains unclear which of the many approaches we have discussed for studying the early development of sidechain interactions will make the key contributions, but all of them should be borne in mind in trying to piece together a picture of this very important aspect of folding. References 1. Kim, P.S. & Baldwin, R.L. (1990). Intermediates in the folding reactions of small proteins. Annu. Rev. Biochem. 59, 631–660. 2. Dill, K.A., Fiebig, K.M. & Chan, H.S. (1993). Cooperativity in proteinfolding kinetics. Proc. Natl Acad. Sci. U.S.A. 90, 1942–1946. 3. Fersht, A.R., Matouschek, A., Sancho, J., Serrano, L. & Vuilleumier, S. (1992). Pathway of protein folding. Faraday Discuss. 93, 183–193. 4. Englander, S.W. & Mayne, L. (1992) Protein folding studied using hydrogen-exchange labelling and two-dimensional NMR. Annu. Rev. Biophys. Biomol. Struct. 21, 243–265. 5. Chaffotte, A.F., Guillou, Y. & Goldberg, M.E. (1992). Kinetic resolution of peptide bond and sidechain far-UV circular dichroism during the folding of hen egg white lysozyme. Biochemistry 31, 9694–9703. 6. Kuwajima, K., Garvey, E.P., Finn, B.E., Matthews, C.R. & Sugai, S. (1991). Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy. Biochemistry 30, 7693–7703. 7. Itzhaki, L.S., Evans, P.A., Dobson, C.M. & Radford, S.E. (1994). Tertiary interactions in the folding pathway of hen lysozyme: kinetic studies using fluorescent probes. Biochemistry 33, 5212–5220. 8. Khorasanizadeh, S., Peters, I.D., Butt, T.R. & Roder, H. (1993). Folding
Review Sidechain interactions in early folding intermediates Engelhard and Evans
9. 10. 11.
12. 13.
14.
15.
16. 17.
18.
19.
20.
21.
22. 23. 24. 25. 26. 27. 28.
29.
30. 31.
and stability of a tryptophan-containing mutant of ubiquitin. Biochemistry 32, 7054–7063. Eftink, M.R. (1991). Fluorescence techniques for studying protein structure. Methods Biochem. Anal. 35, 127–207. Garvey, E.P., Swank, K. & Matthews, C.R. (1989). A hydrophobic cluster forms early in the folding of dihydrofolate reductase. Proteins 6, 259–266. Smith, C.J., Clarke, A.R., Chia, W.N., Irons, W.L., Atkinson, T. & Holbrook, J.J. (1991). Detection and characterization of intermediates in the folding of large proteins by the use of genetically inserted tryptophan probes. Biochemistry 30, 1028–1036. Radford, S.E., Dobson, C.M. & Evans, P.A. (1992). The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature 358, 302–307. Ballery, N., Desmadril, M., Minard, P. & Yon, J.M. (1993) Characterization of an intermediate in the folding pathway of phosphoglycerate kinase – chemical reactivity of genetically introduced cysteinyl residues during the folding process. Biochemistry 32, 708–714. Svensson, M., et al., & Carlsson, U. (1995). Mapping the folding intermediate of human carbonic anhydrase II: probing substructure by chemical reactivity and spin and fluorescence labelling of engineered cysteine residues. Biochemistry 34, 8606–8620. Jones, B.E., Beechem, J.M. & Matthews, C.R. (1995). Local and global dynamics during the folding of Escherichia coli dihydrofolate reductase by time-resolved fluorescence spectroscopy. Biochemistry 34, 1867–1877. Semisotnov, G.V., Rodionova, N.A., Kutyshenko, V.P., Ebert, B., Blanck, J. & Ptitsyn, O. (1987). Sequential mechanism of refolding of carbonic anhydrase B. FEBS Lett. 224, 9–13. Jennings, P.A., Finn, B.E., Jones, B.E. & Matthews, C.R. (1993). Reexamination of the folding mechanism of dihydrofolate reductase from Escherichia coli: verification and refinement of a four channel model. Biochemistry 32, 3783–3789. Kuwajima, K., Mitani, M. & Sugai, S. (1989). Characterization of the critical state in protein folding – effects of guanidine-hydrochloride and specific Ca2+ binding on the folding kinetics of alpha-lactalbumin. J. Mol. Biol. 206, 547–561. Jones, B.E., Jennings, P.A., Pierre, R.A. & Matthews, C.R. (1994). Development of nonpolar surfaces in the folding of Escherichia coli dihydrofolate reductase detected by 1-anilinonaphthalene-8-sulfonate binding. Biochemistry 33, 15250–15258. Semisotnov, G.V., Rodionova, N.A., Razgulyaev, O.I., Uversky, V.N., Gripas, A.F. & Gilmanshin, R.I. (1991). Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers 31, 119–128. Engelhard, M. & Evans, P.A. (1995). Kinetics of interaction of partially folded proteins with a hydrophobic dye: evidence that molten globule character is maximal in early folding intermediates. Protein Sci. 4, 1553–1562. Ptitsyn, O.B., Pain, R.H., Semisotnov, G.V., Zerovnik, E. & Razgulyaev, O.I. (1990). Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 262, 20–24. Lattman, E.E. (1994). Small angle scattering studies of protein folding. Curr. Opin. Struct. Biol. 4, 87–92. Feng, H.P. & Widom, J. (1995). Kinetics of compaction during lysozyme refolding studied by continuous-flow quasielastic light scattering. Biochemistry 33, 13382–13390. Frieden, C., Hoeltzli, S.D. & Ropson, I.J. (1993). NMR and protein folding: equilibrium and stopped-flow studies. Protein Sci. 2, 2007–2014. Balbach, J., Forge, V., Vannuland, N.A.J., Winder, S.L., Hore, P.J. & Dobson, C.M. (1995). Following protein-folding in real-time using NMR-spectroscopy. Nature Struct. Biol. 2, 865–870. Creighton, T.E. (1992). Protein folding pathways determined using disulphide bonds. Bioessays 14, 195–199. Van Mierlo, C.P.M., Darby, N.J., Keeler, J., Neuhaus, D. & Creighton, T.E. (1993). Partially folded conformation of the (30–51) intermediate in the disulphide folding pathway of bovine pancreatic trypsin inhibitor. J. Mol. Biol. 229, 1125–1146. Kawata, Y., Hamaguchi, K. (1991). Use of fluorescence energy transfer to characterise the compactness of the constant fragment of an immunoglobulin light chain in the early stage of folding. Biochemistry 30, 4367–4373. Amir, D., Krausz, S. & Haas, E. (1992). Detection of local structures in reduced unfolded bovine pancreatic trypsin-inhibitor. Proteins 13, 162–173. Matouschek, A., Serrano, L. & Fersht, A.R. (1992). The folding of an
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enzyme IV. Structure of an intermediate in the refolding of barnase analysed by a protein engineering procedure. J. Mol. Biol. 224, 819–835. 32. Horovitz, A. & Fersht, A.R. (1990). Strategy for analysing the co-operativity of intramolecular interactions in peptides and proteins. J. Mol. Biol. 214, 613–617.
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Experimental investigation of sidechain interactions in early folding intermediates Markus Engelhard and Philip A Evans Kinetic studies of folding sometimes reveal very rapid spectroscopic changes that may indicate the population of intermediates, but it is difficult to elucidate in detail the nature of the interactions involved. In this review, we focus on one important aspect of this problem: how to probe the nature and extent of clustering of hydrophobic sidechains. As the information obtainable from different experimental approaches is outlined, it becomes clear that a combination of methods is likely to be necessary to build up a reasonable picture of early folding events. Address: Department of Biochemistry and Cambridge Centre for Molecular Recognition, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK. E-mail addresses: Markus Engelhard, [email protected]; Philip A Evans, [email protected] Electronic identifier: 1359-0278-001-R0031 Folding & Design 01 Apr 1996, 1:R31–R37 © Current Biology Ltd ISSN 1359-0278
Introduction
Protein folding involves both ordering of the backbone into specific, often hydrogen-bonded, structural elements and packing together of sidechains, especially non-polar groups driven to cluster by the hydrophobic effect. Although these are highly interdependent in native protein structures, generalized mechanistic models tend to emphasize the role of one or other aspect in initiating productive folding. The framework model, for example, suggests that elements of locally ordered backbone structure develop initially and then associate to form a tertiary structure [1]. In contrast, the hydrophobic collapse model suggests an overall condensation of the chain driven by clustering of non-polar groups and organization of the backbone within this collapsed state to form the native structure [2]. There is a clear need for detailed experimental evidence to resolve the issue of what really happens in the early stages of folding. Intermediates have been detected experimentally in the folding of many proteins and methods including protein engineering and pulsed hydrogen exchange labelling have been developed for their detailed characterization [3,4]. In the main, intermediates that have been mapped out in detail have turned out to be rather highly organized, with substantial native-like structure, and probably represent relatively late stages in folding. Earlier intermediates, which might tell us more about how folding begins,
remain more enigmatic. Some events are too fast to be studied with current methodologies, but relatively early intermediates, formed within the dead time of a stoppedflow experiment, for example, are often detectable, and understanding their nature would be an important step forward. The problem is to find experiments to characterize species that may be structurally highly heterogeneous and far from native-like. Probes of backbone folding are quite well established. Circular dichroism (CD) can provide reasonable estimates of secondary structure content and can be applied in stopped-flow studies, though it cannot tell us which residues are actually involved [5,6]. More detailed mapping of secondary structures may be possible through hydrogen exchange labelling or protein engineering, although structural heterogeneity and limited stability are problematic for both methods. The problem we are concerned with in this review is the complementary one of how to investigate the nature and extent of sidechain interactions in early intermediates. A wide variety of spectroscopic, and other, probes have been used to follow folding reactions and most are potentially sensitive to changes in the environment of one or more sidechains. Thus, detection of any change at a rate faster than overall folding is commonly interpreted in terms of population of a folding intermediate. However, most methods actually provide very little detail about the interactions involved. In looking at the earliest detectable events in a stopped-flow experiment, it may be difficult to distinguish between simple changes in the properties of a probe as its solvent environment is changed, which might be of no consequence for the folding mechanism, and potentially more significant events such as local or global hydrophobic collapse. Actually mapping the sidechain clusters is a much more difficult problem, but one that must be addressed if we are to establish not just when condensation happens but how important an aspect of folding it really is. For example, a non-specific ‘stickiness’ of hydrophobic groups might be quite incidental or even a positive obstacle to be overcome in folding. Global collapse, on the other hand, might help to ensure efficient folding by favouring an approximation of the native chain topology at an early stage in the pathway. We need detailed structural information to gain a better perspective on these possibilities. Our focus in this review is therefore on the information content of the various methods available for looking at sidechain interactions in the early stages of folding.
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Probe accessibility methods
The presumed driving force for sidechain clustering, diminished interaction with water, also provides one strategy for its detection: if one participating group is a suitable probe, such as a fluorophore, it may be possible to detect the change in its solvent accessibility. Tryptophan and tyrosine residues are intrinsic fluorophores and their emission intensities, which are sensitive to interactions both with solvent and with other sidechains, often change markedly during folding. Intensity changes are most often used to monitor formation of the native state, but additional kinetic phases associated with faster processes are sometimes apparent. Changes occurring within the dead time of a stopped-flow experiment can also be measured, although this depends on careful extrapolation of the fluorescence of the starting denatured state to the conditions of refolding [7,8]. The sensitivity of fluorescence intensity to a variety of environmental factors means that it should never be interpreted in specific structural terms [9], but other fluorescence parameters may provide a more straightforward reflection of solvent accessibility. The wavelength of maximal emission, lmax, of tryptophan (but not tyrosine) residues is influenced by polar interactions in the excited state which are effective in water but typically much less so in the hydrophobic protein interior. Hydrophobic contacts formed by the indole ring can thus be detected by a blue shift in its emission spectrum [9]. This has not yet been widely exploited in kinetic studies because most stopped-flow fluorescence set-ups simply integrate total emission, rather than generating spectra. With the increased availability of diode array detection to provide wavelength-dependent data, it seems likely that the method will be explored more widely in the future.
tions cannot usually be resolved and assigned. A more global picture of the distribution of clusters may be obtainable by using protein engineering to introduce (and, if necessary, delete) tryptophans at different places in the structure and compare the time-dependent accessibility changes of the different probes [8,11]. These experiments depend on the kinetics and mechanism of folding not being seriously perturbed by the mutations. This is not always easy to achieve, because the fused ring system of tryptophan is seldom a very conservative substitution. Careful controls are needed, therefore, in interpreting these experiments. Other probe accessibility methods have also been used, notably chemical labelling methods. A potential advantage here is that if a reaction is effectively irreversible, analytical methods can subsequently be used to characterize the pattern of reactivity of multiple probes within a single molecule. This idea is essentially the same as that of hydrogen exchange labelling, but in that case most of the probes are backbone NHs, so that only indirect implications about sidechain interactions are obtained. Important exceptions to this are the indole NHs of tryptophan residues, protection of which has indeed been detected in some early folding intermediates [12]. Other labelling methods are based on reactions of sidechain functional groups, most notably the thiol group of cysteine, which can react very rapidly as a nucleophile. Selective mutation of different sidechains to cysteine has been used as a way of introducing this probe into proteins and differential reactivity has been observed in both kinetic and equilibrium intermediates and has been interpreted in terms of accessibility to the labelling reagent [13,14]. Probe mobility methods
Another relatively simple way to obtain a more direct measure of fluorophore accessibility uses small molecule co-solutes such as iodide or acrylamide as quenchers [9]. As quenching is a contact interaction, it will be diminished if the accessibility of a fluorophore is restricted by hydrophobic clustering, as has indeed been detected in early folding intermediates of some proteins [7,10]. However, the extent of quenching may also vary with changes in fluorescence lifetime and, if the quencher is charged, in the electrostatic environment, so this provides only a qualitative measure of accessibility changes during folding. As with any additive to the medium, it is important to design experiments to ensure that the quencher does not perturb the folding kinetics [7]. In proteins having only a single tryptophan, these methods can potentially provide specific information about the involvement of that residue in hydrophobic clusters; where there are multiple fluorophores, only more general conclusions can be drawn, as individual contribu-
Another approach to the detection of sidechain interactions depends on the reduction in mobility that clustering entails. Various spectroscopic methods are available that are sensitive to molecular motions, though not all are immediately applicable to proteins. Intrinsic fluorescence is certainly influenced by dynamics, but these effects cannot be distinguished from other factors in simple steady-state fluorescence intensity measurements. Much more detailed information is obtainable from measurements of fluorescence decay rates and depolarization, for which simple models are available that allow interpretation in terms of sidechain dynamics. This approach is experimentally much more demanding than steady-state fluorescence, but it has recently been shown to be capable of providing a detailed reflection of the gradual immobilization of tryptophan sidechains as folding progresses [15]. Other techniques that have been used to detect mobility changes during refolding have used chemical modification to introduce a suitable reporter group. In particular, elec-
Review Sidechain interactions in early folding intermediates Engelhard and Evans
tron paramagnetic resonance (EPR) can be used to monitor the mobility of spin labels [16]. The relaxation rates of the electron spin are very sensitive to dynamics and it has been shown that loss of signal, corresponding to a broadening of the spectrum, provides a good probe for immobilization. In early applications, probes were introduced by non-selective sidechain modification [16], but the method can be refined by using site-directed mutagenesis to introduce reactive residues such as cysteine at a range of sites in the sequence, allowing spin-labels to be introduced at strategic places in the structure [14]. The spin labels used in these experiments tend to be substantial hydrophobic groups themselves, so that a potential complication arises from their ability to participate directly in hydrophobic clustering interactions. The principal problem with mobility-based approaches, even more than with those dependent on accessibility changes, is that it is hard to gauge the extent to which the effects observed reflect significant folding events, as opposed to strictly local changes in conformational freedom. Introduction of probes at a range of sites goes some way to addressing this problem, but it is still difficult to know how extensive and how fixed a set of interactions is implied by a given change in the experimental parameters observed. Interaction with extrinsic hydrophobic probes
An alternative to monitoring the behaviour of probes within a folding molecule is to employ external agents capable of interacting with the protein in a conformationdependent manner. An obvious example is the use of ligands of the native state to detect the formation of a functional binding site. In most cases, this effectively measures the formation of the native state [7,17], although relatively localized binding sites, such as metal-binding loops, may become organized at an earlier stage in folding [18]. A different type of extrinsic probe that has achieved popularity in folding studies is exemplified by 8-anilino-1naphthalenesulphonate (ANS), a hydrophobic fluorescent dye [16,19]. Its fluorescence is effectively quenched in aqueous solution, but the quantum yield increases sharply on binding to a hydrophobic surface. It has been found that non-polar groups in unfolded proteins are too diffuse to allow significant interaction with ANS, whereas in fully folded proteins, unless they have hydrophobic binding regions for functional reasons, the non-polar groups are inaccessible to the dye as they are sequestered in the interior. In contrast, some intermediates can interact very effectively, causing large enhancements in ANS fluorescence, presumably because non-polar groups are now clustered but with sufficient exposure to the outside or with sufficient flexibility in the structure to allow access to the dye. This remarkable kind of specificity has led to widespread applications for the method in identifying partially folded states of proteins [7,16,19,20].
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In kinetic studies of folding, ANS fluorescence has generally been investigated simply by including the dye in the refolding buffer [20]. Time courses observed in this way for refolding of several proteins were qualitatively remarkably similar: a moderate dead-time fluorescence increase was followed by a further, slower, rise before the signal gradually returned to a low level as the protein reached its native state. These results were interpreted on the basis of two key assumptions: first, that the dye bound instantaneously, so that any time-dependent effects could be assumed simply to be reporting conformational changes in the protein; second, that interaction with the dye would significantly perturb neither the folding pathway nor its kinetics. Unfortunately, it turns out that neither assumption is always valid: detailed studies of two proteins revealed that some kinetic phases observable through ANS fluorescence enhancement are actually due to the formation or reorganization of dye–protein complexes, rather than simply reflecting the progress of folding [21]. Further, it was clear that ANS could indeed profoundly alter the folding kinetics, presumably by stabilizing intermediates to which it binds. These problems can be alleviated by using a pulse method, where the ANS is introduced only after folding has been allowed to proceed for a given time, and folding curves are reconstructed by plotting the initial fluorescence then observed, as a function of the folding time [21]. Additional information from ANS binding is obtainable by applying time-resolved fluorescence measurements which can provide insight into the mobility of the bound dye molecule [15]. Global compactness
The techniques discussed so far are relatively straightforward to apply and can provide good evidence for some kind of hydrophobic clustering. They do not, however, provide information as to the extent of clustering and cannot be used to map the residues involved. There are various ways to estimate the extent of global condensation of a protein molecule, but they are not simple spectroscopic methods and their application to kinetic intermediates has been a demanding problem. In exceptional cases where intermediates are very long-lived, it has been possible to use gel permeation chromatography to demonstrate their compact structures [22], but methods of this kind are clearly inappropriate for the ephemeral early intermediates that are commonly encountered in the folding of small proteins. Small-angle X-ray scattering is a powerful method for studying the compactness of macromolecules and has been applied to several studies of equilibrium partially folded states, but kinetic applications are in their infancy. The principal limitation is the low sensitivity of the technique, but improved radiation sources and experimental designs promise much in the future [23]. Quasielastic light scattering can be used to study translational mobility, and
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hence compactness, during folding, although again this method is in the early stages of development: good evidence for almost native-like condensation of a partially folded state of lysozyme was obtained, although a long dead time (around 1 s) meant that this corresponded to a relatively late intermediate [24]. There seems to be no fundamental barrier to extending either of these approaches to the point where following the kinetics of compaction would be a realistic possibility. Proximity determination methods
If we actually want to map the structure present in a folding intermediate, the most direct way is to determine approximate average distances between specific residues. This is, of course, the basis of the standard NMR method for solution structure determination, where NOEs can provide a wealth of distance restraints from which detailed structures can be calculated. Progress is being made in applying NMR to kinetic folding intermediates, although applications are likely to be limited to relatively long-lived species [25,26]. Other ways to derive proximity information are based on much more limited numbers of specific probes. The trapping of disulphide intermediates in the oxidative refolding of proteins is a classic example. Formation of a specific disulphide with sufficient population to allow it to be trapped requires it to be stabilized by cooperation with non-covalent interactions among sidechains around the participating cysteines, so this is effectively a direct means of detecting hydrophobic clusters [27]. Further, intermediates with subsets of disulphides can be trapped and isolated, or modelled through protein engineering, so that their conformations can be studied in detail by, for example, NMR [28]. Oxidative refolding is typically much slower than that of small globular proteins lacking disulphides, but this does not mean that the mechanisms involved are fundamentally different, only that events tend to be slowed down by the thiol chemistry involved. This approach is potentially very powerful for studying disulphide-containing proteins; it has been applied in great detail in one case, but it is to be hoped that it can be extended to other systems and that the conclusions drawn can be integrated more fully with those deriving from other techniques. Another phenomenon that can provide proximity information is fluorescence energy transfer. This idea has been used in a general way by introducing dansyl groups nonselectively and monitoring the development of their emissions when tryptophans in the protein were excited: the efficiency of energy transfer was observed to increase markedly during folding, presumably reflecting shorter average distances between the donor and acceptor groups [16]. In some cases, it is possible to generate a specific donor/acceptor pair, where unique residues are present, or
can be genetically introduced, that are suitable for modification. For example, energy transfer between a single tryptophan and a single labelled cysteine allowed detection of an early compact intermediate in the folding of an immunoglobulin light chain [29]. Measurement of individual average distances requires such unique donor/acceptor pairs and the capability to make time-resolved fluorescence measurements. This idea has been applied to a variety of equilibrium non-native states of one protein, in which different probe combinations could be used to derive distance information, allowing conclusions about the extent of condensation to be drawn [30]. Protein engineering
The principal limitation of most of the physical and chemical methods discussed above is that they can provide only limited residue-specific information. The protein engineering approach, on the other hand, offers the potential to examine quantitatively how the interactions of any chosen sidechain contribute to the stability of folding intermediates and transition states. A simple detection method such as fluorescence can be used to follow overall folding, without any need to observe the sidechain directly. Applied in a systematic way, this can provide an extraordinarily detailed map of these species [3]. The prerequisite for application of the method is a relatively simple kinetic scheme, in which it can be assumed that the native and unfolded states, sometimes a metastable intermediate, and the transition states in between are all well defined and structurally independent of the solution conditions. The idea then is that selectively mutating an individual sidechain will perturb the relative stabilities of the various states in a way that depends on the extent to which the residue concerned is involved in the folded structure in each case. These free energy perturbations can be measured from a combination of equilibrium unfolding and kinetic unfolding/refolding data. This approach has been applied intensively to a small number of systems, allowing detailed mapping of the interactions present in the transition states. In one case, barnase, the presence of a well populated intermediate was also inferred and its structure mapped out [31]. In all of these instances the structure present appeared to be native-like but ‘weakened’ to varying degrees. It is possible to check for non-native interactions through analysis of so-called double mutant cycles [32], and where this has been done the assumption of native-like structure has largely been validated. The information deriving from these studies has been uniquely detailed and the method has other advantages in that, for example, it provides direct information as to the kinetic significance of the intermediates concerned. It is clear, however, that the species so far investigated in this way have rather highly developed structure, at least
Review Sidechain interactions in early folding intermediates Engelhard and Evans
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niques suggest a series of different partially folded forms, it would be difficult to obtain comparably detailed information; at the very least, it would be necessary to use different detection methods to pick out particular intermediates.
locally, and hence represent relatively advanced stages of folding. In principle, it should be possible to extend the method to earlier intermediates, but there are a number of potential difficulties and it will be interesting to see how effectively it can be applied to intermediates that other techniques suggest are not highly native-like. The use of a single spectroscopic probe to monitor transition between unfolded and fully folded states has been an attractive aspect of the applications so far, as it allows simple and sensitive methods to be applied. The data thus obtained are sufficient only if a maximum of one populated intermediate is assumed, however. In cases where other tech-
Detailed interpretation of protein engineering experiments depends on two key assumptions: any folding interactions present should be native-like and the mutations made should be non-disruptive of the structures of the species involved. These points will both have to be examined carefully in extending the approach to early interme-
Figure 1
I
unfolded state
II
native state
= tryptophan = sidechain contributing to the principal hydrophobic core
Trp fluorescence a
= covalently linked fluorophore
Intensity changeb
?
?
?
Accessibilityc Mobility ANS bindingd
X
?
X
X
X
Compactness Energy transfere Native functional properties
A hypothetical folding pathway in which two intermediates — I, involving local clustering of groups of residues, and II, a globally collapsed, molten globule state — are sequentially populated. This is not proposed as a general model for folding, nor would the population of the intermediates necessarily be kinetically resolved, but it serves to illustrate the different aspects of folding that might be detected by some of the probes discussed in the text. aA single tryptophan fluorophore is used as an example of a ‘local’ structural probe. Other such probes responding to accessibility or mobility changes are discussed in the text. bAs fluorescence intensity depends on a variety of environmental factors, changes in each stage of folding are likely,
X
but their magnitudes and directions cannot be easily predicted. cIn this particular hypothetical protein, the Trp sidechain becomes involved in a cluster at an early stage, with consequent accessibility and mobility changes. Detection of such an intermediate, however, is partly a matter of chance — this early intermediate might escape detection by such a probe if the latter were located elsewhere in the sequence. dIt is not clear how extensively structured an intermediate needs to be to bind ANS efficiently — thus it is uncertain whether the early intermediate would be detected. eFluorescence of a covalently linked fluorophore as a result of energy transfer from the single Trp can be used to probe changes in the average separation of the two groups.
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diates. Effects of non-native interactions can be detected through double mutant cycles [32], but it is hard to pin down any particular non-native structure that may be present. This has not proved to be a serious limitation in the applications reported to date, but it remains to be seen whether this will be true in states further from the native structure. Relaxation of folded and partially folded structures in response to mutations has been observed to diminish the consequent free energy perturbations to varying degrees. Again, these effects have not been a serious barrier to application of the method so far, but if the structure of an early intermediate were very flexible, the capacity for rearrangement and compensation for mutations might be correspondingly greater. Thus, in cases where protein engineering suggests that interactions of a particular sidechain are substantially absent in an intermediate, it will be necessary to take care to eliminate the alternative possibility: that the interactions it forms are just not very specific. Conclusions
Many techniques are now available that can contribute to developing our understanding of how sidechains begin to interact in early folding intermediates. They are clearly not all sensitive to structure at the same level; for example, states that bind ANS may nonetheless have highly mobile tryptophan sidechains [15]. Thus, no single method can provide a complete picture and it is clearly desirable that data from a variety of approaches should be assembled before drawing too many firm conclusions in a particular case (see Fig. 1). If our understanding of the overall organization of folding is to benefit significantly from studying this behaviour, we will need a much more detailed picture of the specific interactions present than has hitherto been achieved for early intermediates. Experiments that can provide this information directly are very demanding and it seems unlikely that more than a few approximate distance constraints will be obtainable in any given case. The indirect evidence obtained from protein engineering can potentially provide the solution to this problem, as it has in the characterization of more highly structured species on folding pathways, although there are likely to be a number of difficulties if the set of interactions present has significant flexibility. Perhaps the key to making sense of the data we can obtain from kinetic experiments on early folding intermediates is to draw them together with results from alternative approaches. Equilibrium denatured states and protein fragments provide a very important point of comparison. Many of the same indicators of partial structuring observable in early kinetic intermediates, such as diminished probe accessibility and mobility and ANS binding, are also observed in some of these species and it seems very likely
that they share many similar properties. The big difference, however, is that these equilibrium states can be studied at leisure and are therefore much more amenable to methods such as NMR that are intrinsically slow but can potentially provide a wealth of structural detail. The significance of the other spectroscopic effects should be much clearer in the light of this kind of information. A further point is that the very earliest events in folding are much too fast to be resolved by conventional methods and it may well be the case that studying fragments, where folding is effectively halted at a particular stage for want of the other residues needed for wider structure development, will be the only way to understand what is likely to happen on this timescale. Theoretical methods for predicting folding mechanisms constitute another important complementary approach. There are two broad classes of approach. One is to analyze the native structure and devise an algorithm that will identify aspects of the structure that might be formed at an early stage of folding. These methods necessarily involve a great deal of assumption about the general nature of initiation events, so it is clear that they should be developed closely in parallel with the growth of our understanding deriving from experimental evidence. Other methods actually aim to simulate all or part of the folding pathway, using either a simplified model for the protein molecule or explicit molecular dynamics; in these cases too, comparison with experimental data will be vital to determine the extent to which the models set up are a useful approximation. This field has seen a great deal of activity and it seems certain that there will be major developments in our understanding of the early events in folding in the coming years. It remains unclear which of the many approaches we have discussed for studying the early development of sidechain interactions will make the key contributions, but all of them should be borne in mind in trying to piece together a picture of this very important aspect of folding. References 1. Kim, P.S. & Baldwin, R.L. (1990). Intermediates in the folding reactions of small proteins. Annu. Rev. Biochem. 59, 631–660. 2. Dill, K.A., Fiebig, K.M. & Chan, H.S. (1993). Cooperativity in proteinfolding kinetics. Proc. Natl Acad. Sci. U.S.A. 90, 1942–1946. 3. Fersht, A.R., Matouschek, A., Sancho, J., Serrano, L. & Vuilleumier, S. (1992). Pathway of protein folding. Faraday Discuss. 93, 183–193. 4. Englander, S.W. & Mayne, L. (1992) Protein folding studied using hydrogen-exchange labelling and two-dimensional NMR. Annu. Rev. Biophys. Biomol. Struct. 21, 243–265. 5. Chaffotte, A.F., Guillou, Y. & Goldberg, M.E. (1992). Kinetic resolution of peptide bond and sidechain far-UV circular dichroism during the folding of hen egg white lysozyme. Biochemistry 31, 9694–9703. 6. Kuwajima, K., Garvey, E.P., Finn, B.E., Matthews, C.R. & Sugai, S. (1991). Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy. Biochemistry 30, 7693–7703. 7. Itzhaki, L.S., Evans, P.A., Dobson, C.M. & Radford, S.E. (1994). Tertiary interactions in the folding pathway of hen lysozyme: kinetic studies using fluorescent probes. Biochemistry 33, 5212–5220. 8. Khorasanizadeh, S., Peters, I.D., Butt, T.R. & Roder, H. (1993). Folding
Review Sidechain interactions in early folding intermediates Engelhard and Evans
9. 10. 11.
12. 13.
14.
15.
16. 17.
18.
19.
20.
21.
22. 23. 24. 25. 26. 27. 28.
29.
30. 31.
and stability of a tryptophan-containing mutant of ubiquitin. Biochemistry 32, 7054–7063. Eftink, M.R. (1991). Fluorescence techniques for studying protein structure. Methods Biochem. Anal. 35, 127–207. Garvey, E.P., Swank, K. & Matthews, C.R. (1989). A hydrophobic cluster forms early in the folding of dihydrofolate reductase. Proteins 6, 259–266. Smith, C.J., Clarke, A.R., Chia, W.N., Irons, W.L., Atkinson, T. & Holbrook, J.J. (1991). Detection and characterization of intermediates in the folding of large proteins by the use of genetically inserted tryptophan probes. Biochemistry 30, 1028–1036. Radford, S.E., Dobson, C.M. & Evans, P.A. (1992). The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature 358, 302–307. Ballery, N., Desmadril, M., Minard, P. & Yon, J.M. (1993) Characterization of an intermediate in the folding pathway of phosphoglycerate kinase – chemical reactivity of genetically introduced cysteinyl residues during the folding process. Biochemistry 32, 708–714. Svensson, M., et al., & Carlsson, U. (1995). Mapping the folding intermediate of human carbonic anhydrase II: probing substructure by chemical reactivity and spin and fluorescence labelling of engineered cysteine residues. Biochemistry 34, 8606–8620. Jones, B.E., Beechem, J.M. & Matthews, C.R. (1995). Local and global dynamics during the folding of Escherichia coli dihydrofolate reductase by time-resolved fluorescence spectroscopy. Biochemistry 34, 1867–1877. Semisotnov, G.V., Rodionova, N.A., Kutyshenko, V.P., Ebert, B., Blanck, J. & Ptitsyn, O. (1987). Sequential mechanism of refolding of carbonic anhydrase B. FEBS Lett. 224, 9–13. Jennings, P.A., Finn, B.E., Jones, B.E. & Matthews, C.R. (1993). Reexamination of the folding mechanism of dihydrofolate reductase from Escherichia coli: verification and refinement of a four channel model. Biochemistry 32, 3783–3789. Kuwajima, K., Mitani, M. & Sugai, S. (1989). Characterization of the critical state in protein folding – effects of guanidine-hydrochloride and specific Ca2+ binding on the folding kinetics of alpha-lactalbumin. J. Mol. Biol. 206, 547–561. Jones, B.E., Jennings, P.A., Pierre, R.A. & Matthews, C.R. (1994). Development of nonpolar surfaces in the folding of Escherichia coli dihydrofolate reductase detected by 1-anilinonaphthalene-8-sulfonate binding. Biochemistry 33, 15250–15258. Semisotnov, G.V., Rodionova, N.A., Razgulyaev, O.I., Uversky, V.N., Gripas, A.F. & Gilmanshin, R.I. (1991). Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers 31, 119–128. Engelhard, M. & Evans, P.A. (1995). Kinetics of interaction of partially folded proteins with a hydrophobic dye: evidence that molten globule character is maximal in early folding intermediates. Protein Sci. 4, 1553–1562. Ptitsyn, O.B., Pain, R.H., Semisotnov, G.V., Zerovnik, E. & Razgulyaev, O.I. (1990). Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 262, 20–24. Lattman, E.E. (1994). Small angle scattering studies of protein folding. Curr. Opin. Struct. Biol. 4, 87–92. Feng, H.P. & Widom, J. (1995). Kinetics of compaction during lysozyme refolding studied by continuous-flow quasielastic light scattering. Biochemistry 33, 13382–13390. Frieden, C., Hoeltzli, S.D. & Ropson, I.J. (1993). NMR and protein folding: equilibrium and stopped-flow studies. Protein Sci. 2, 2007–2014. Balbach, J., Forge, V., Vannuland, N.A.J., Winder, S.L., Hore, P.J. & Dobson, C.M. (1995). Following protein-folding in real-time using NMR-spectroscopy. Nature Struct. Biol. 2, 865–870. Creighton, T.E. (1992). Protein folding pathways determined using disulphide bonds. Bioessays 14, 195–199. Van Mierlo, C.P.M., Darby, N.J., Keeler, J., Neuhaus, D. & Creighton, T.E. (1993). Partially folded conformation of the (30–51) intermediate in the disulphide folding pathway of bovine pancreatic trypsin inhibitor. J. Mol. Biol. 229, 1125–1146. Kawata, Y., Hamaguchi, K. (1991). Use of fluorescence energy transfer to characterise the compactness of the constant fragment of an immunoglobulin light chain in the early stage of folding. Biochemistry 30, 4367–4373. Amir, D., Krausz, S. & Haas, E. (1992). Detection of local structures in reduced unfolded bovine pancreatic trypsin-inhibitor. Proteins 13, 162–173. Matouschek, A., Serrano, L. & Fersht, A.R. (1992). The folding of an
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enzyme IV. Structure of an intermediate in the refolding of barnase analysed by a protein engineering procedure. J. Mol. Biol. 224, 819–835. 32. Horovitz, A. & Fersht, A.R. (1990). Strategy for analysing the co-operativity of intramolecular interactions in peptides and proteins. J. Mol. Biol. 214, 613–617.