Antagonism, non-native interactions and non-two-state folding in S6 revealed by double-mutant cycle analysis

Daniel Otzen

Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark E-mail: dao{at}bio.aau.dk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
When folding to the native state N in the presence of salt, the apparent two-state folder S6 transiently forms a transient off-pathway state C with substantial secondary and tertiary structure. Fifteen double mutant cycles were analysed to compare side-chain interaction energies {Delta}{Delta}Gint in C, N and TS (the transition state between N and the denatured state). The kinetic signatures of these destabilizing mutants suggest folding scenarios involving unfolding intermediates and even alternative unfolding pathways. However, restricting the kinetic data to linear parts of the chevron plot allows reliable extrapolation to zero molar denaturant of rate constants of folding, unfolding and misfolding. Side-chain interactions appear to contribute to the stability of C, but in a substantially non-native environment, as shown by changes in the sign of {Delta}{Delta}Gint between C and N. Remarkably, there appear to be significant (0.7–2 kcal/mol) antagonistic interactions between the two residues Leu30 and Leu75 in N and TS, which may be linked to subtle structural changes seen in the crystal structures of the mutants. A small number of overlapping residues are involved in these kinds of antagonistic interactions in N, TS and C, suggesting that repulsive interactions are coded into the protein topology whether the protein folds or misfolds. Destabilizing double mutants indicate that apparent two-state folders can be induced to behave in more complex ways provided that the native state is suitably destabilized.

Keywords: chevron plots/crystallography/interaction energies/kinetics/mutants


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The energy landscape model has been proposed as a way to view the directed conformational search that occurs when a protein folds (Onuchic et al., 1996Go; Dill, 1999Go). An attractive feature of this model is that partially structured states are not intrinsically ‘on-’ or ‘off-pathway’, but rather represent local energy minima which may direct but also retard the protein in its quest for the most stable state. Detailed characterization of these states may shed more light on the principles governing a protein's exploration of the energy landscape. An example of such a partially folded state is provided by the 101-residue ribosomal protein S6 from Thermus thermophilus. In the absence of salt, S6 folds without any detectable intermediates between the denatured (D) and native (N) states (Otzen et al., 1999Go; Otzen and Oliveberg, 2002Go). As such, it is included as a ‘model two-state protein’ in a recent comprehensive analysis (Maxwell et al., 2005Go). Nevertheless, this apparent simplicity of behaviour does not survive a closer examination. First, for most S6 mutants there is considerable curvature in the unfolding limb of the chevron plot (logarithm of the observed rate constant kobs versus denaturant concentration), which can be interpreted either as the formation of an unfolding intermediate, plasticity in the native state or transition state movement (Otzen et al., 1999Go; Otzen and Oliveberg, 2002Go). Strong evidence for the formation of an unfolding intermediate is provided in the present paper. Second, the presence of stabilizing salts such as sodium sulfate leads to the transient accumulation of a collapsed state C, which slows folding by several orders of magnitude (Otzen and Oliveberg, 1999Go). C is almost native-like in its compactness and degree of secondary structure, but has either to unfold back to D or to undergo extensive rearrangements, including a small but significant degree of expansion (Otzen, 2005Go) to reach N. A protein engineering analysis of C using the f value approach suggested that the residues fall into two classes, one of which retains a significant degree of secondary structure, while the other class is essentially unstructured (Otzen and Oliveberg, 1999Go). However, the analysis is complicated by the fact that C is not necessarily intermediate between D and N in its structural consolidation, making {Phi} values difficult to interpret. In an attempt to obtain a more reliable estimate of side-chain interactions of C and relate them to their interactions in the native state and transition state of folding, double-mutant cycles of 15 residue pairs have been carried out. In this approach (Carter et al., 1984Go; Horovitz and Fersht, 1992Go; Horovitz, 1996Go), two residues are mutated singly and pairwise. If the two residues are coupled in energy terms, there will be a coupling energy {Delta}{Delta}Gint between them, calculated as

(1)
where the {Delta}{Delta}G values represent energy changes relative to wild-type and the superscripts refer to the double-mutant (1 + 2) and the single mutants (1 and 2). A negative value for {Delta}{Delta}Gint indicates that there is an attractive interaction energy between the two mutated residues. The virtue of double-mutant cycles is that they provide a simple way to estimate the strength of pairwise residue interactions in structurally well-defined states (even if these states are non-native), since the surrounding side-chain interaction energies and reorganization energies effectively cancel out in Equation 1. In addition to their use in studying cooperativity and antagonism in enzyme mechanisms (Carter et al., 1984Go; Wells, 1990Go; Mildvan, 2004Go), double-mutant cycles have been used to measure intramolecular side-chain contacts in intermediates and transition states of folding (Horovitz et al., 1990Go; Itzhaki et al., 1995Go; Parker et al., 1996Go) and intermolecular contacts (Hidalgo and MacKinnon, 1995Go; Schreiber and Fersht, 1995Go; Quiram et al., 1999Go).

When two residues are known to be in contact with each other, the extent of coupling between them generally reflects direct contacts and decreases with distance. In the absence of structural information, coupling energies should be approached with caution, since weak coupling energies may arise from non-isotropic structural adjustments to the mutation. Strong coupling energies may reflect indirect (primarily electrostatic) interactions which can propagate through the low dielectric protein interiors (Jackson and Fersht, 1993Go; Otzen and Fersht, 1999Go). In such cases, the existence of specific structural interactions should be underpinned by detailed mutagenic analysis of the residues surrounding the electrostatic pair (Hidalgo and MacKinnon, 1995Go).

All of the double mutant cycles in this study probe hydrophobic contacts present in the native state, which should minimize complications from long-range interactions. The data suggest that extensive side-chain contacts are established in C, although they appear to have non-native character. Furthermore, an unusual degree of side-chain antagonism is revealed in N, C and TS, centred around a small number of ‘hot-spot’ residues which only partially overlap between the different states. Finally, the data provide evidence not only for a conventional unfolding intermediate but also for parallel pathways of unfolding of different S6 mutants, illustrating how the destabilization of a protein can reveal multiple routes between the denatured and native states in the energy landscape.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Double mutants were constructed, expressed and purified and their rates of unfolding and refolding were determined by fluorescence stopped-flow kinetics as described (Otzen and Oliveberg, 1999Go; Otzen et al., 1999Go). The mutants LA75 and LA30/LA75 were crystallized in 0.2 M sodium citrate, 0.1 M Tris–HCl pH 8.5 and 17.5% (v/v) (LA75) or 25% (LA30/LA75) PEG400 and their crystal structures elucidated as described by molecular replacement (Otzen et al., 1999Go). Notably, the mutant sequences were 100% correct and the crystal structures of LA75 and LA30/LA75 were consistent with the expected mutations. The crystal structures of S6 wild-type (1RIS) and LA30 (1LOU) are available from previous work (Lindahl et al., 1994Go; Otzen et al., 1999Go). Cavity sizes were calculated using the program VOIDOO with a probe radius of 1.2 Å. All experiments were carried out in 50 mM MES pH 6.3. Temperatures were kept to 25°C unless stated otherwise. The coordinates for the structures have been deposited in the PDB database as 2BVZ (LA75) and 2BXJ (LA30/LA75). Details of data collection and refinement statistics are given in Table I.


View this table:
[in this window]
[in a new window]
 
Table I. Crystallographic data for the mutants LA75 and LA30/LA75

 
Data analysis

The following four folding schemes are used, where D, I, C and N are the denatured, intermediate, collapsed and native states, respectively, kf is the rate constant of refolding, ku and k*u are rate constants of unfolding and KI = [I]/N] and KC = [C]/D].

Two-state folding: Three-state folding where the intermediate only accumulates transiently during unfolding:



View larger version (7K):
[in this window]
[in a new window]
 
Scheme 1.
 


View larger version (5K):
[in this window]
[in a new window]
 
Scheme 2.
 
Three-state folding with alternative unfolding route:



View larger version (12K):
[in this window]
[in a new window]
 
Scheme 3.
 
Refolding with a collapsed-state dead-end:



View larger version (8K):
[in this window]
[in a new window]
 
Scheme 4.
 
Kinetic data in the absence of salt were fitted to the two-state model (Scheme 1), in which logarithmic refolding and unfolding rates depend linearly on [GdmCl] (Tanford, 1970Go):

(2)
where kobs is the measured rate constant, G is [GdmCl], and are refolding and unfolding rates in water and mf and mu are the linear dependences of logkf and logku on [GdmCl]. For this analysis, unfolding rate constants outside the linear region of the unfolding limb of the chevron plot are excluded (see Results).

To include the whole unfolding limb for all mutants except IA8/LA79, data were analysed according to Scheme 2 with the following equation:

(3)
where mI is the linear dependence of logKI on [GdmCl].

For the mutant IA8/LA79, Scheme 3 was applied with the accompanying equation:

(4)
where mu* is the linear dependence of logku* on [GdmCl].

Data on the collapsed state C were obtained from refolding rates in 1 M Na2SO4, fitted to the following equation based on Scheme 4 (Otzen and Oliveberg, 1999Go):

(5)
where mC is the linear dependence of logKC on [GdmCl].

Interaction energies were calculated as follows, where the superscripts refer to the single mutants (1 and 2) and the double mutant (1 + 2) (Horovitz and Fersht, 1990Go, 1992Go):

Interactions in terms of activation energies of refolding:

(6)
Interactions in terms of activation energies of unfolding:

(7)
Interactions in the native state N:

(8)
Interactions in the compact state C:

(9)


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Selection of mutated residues

To probe the strength of individual side-chain interactions, 15 pairs of mutants were selected, whose side chains are within contact-distance of each other in the crystal structure (Lindahl et al., 1994Go) (Table II). For example, the six first double mutations in Table II form a network of interlocking hydrophobic contacts involving seven different side chains (Figure 1). All of the mutated residues are largely buried and form part of the hydrophobic core of S6 or, in the case of Glu22, a hydrogen-bonding network with Arg82 and Asn84. The double mutant EQ22/VA85 was constructed because both single mutations stabilize C to a large extent and lead to the opening of an alternative folding route to N (Otzen and Oliveberg, 1999Go). As a consequence, we have termed them ‘gatekeeper residues’, which we define as residues that impede misfolding without necessarily stabilizing the folded state. Glu22 and Val85 are close to each other in N, so a degree of interaction is also expected in this state. For each mutant, refolding and unfolding rates as a function of denaturant concentration were measured in the absence of salt to characterize the effect of the mutations on the stabilities of the native state and transition state (TS). Refolding rates measured in the presence of 1 M Na2SO4 enable us to determine the stability of the collapsed state C (Table III). Because of the extreme destabilization observed in many of the double mutants, the concentration range over which the protein was predominantly native was too narrow for equilibrium denaturation experiments to provide reliable native-state baselines; therefore, this work was restricted to an analysis based on kinetic data.


View this table:
[in this window]
[in a new window]
 
Table II. Kinetics for double mutants analysed according to a three-state model (Scheme 2, Equation 3)

 


View larger version (70K):
[in this window]
[in a new window]
 
Fig. 1. Structure of S6, highlighting contacts between residues probed by double mutant cycles. The mutated side chains are grouped as three sets of residues whose pairwise interactions are probed by double mutant cycles (cf. Table II). Residues Leu6, Ile8, Leu 26, Leu30, Leu 75, Leu79 and Val90 are in dark grey, Ala35 and Met67 are in light grey and Glu22 and Val85 are in white.

 

View this table:
[in this window]
[in a new window]
 
Table III. Kinetic data for the double mutants analysed in this study and their corresponding single mutantsa

 
The double mutants show clear deviation from simple two-state behaviour, including behaviour consistent with unfolding intermediates and parallel unfolding pathways

Although S6 wild-type shows a classical V-shaped chevron plot (logkobs versus denaturant concentration), several destabilizing mutations have been shown previously to give rise to substantial curvature in the plot's unfolding limb at high denaturant concentrations. We have interpreted this as movement of the transition state in response to changing solvent conditions (Otzen et al., 1999Go), although the data are also compatible with a model in which the native state becomes more ‘plastic’ at higher denaturant concentrations and therefore partially unfolds in the dead time of the stopped-flow experiments prior to unfolding proper (Otzen and Oliveberg, 2002Go); however, an unfolding intermediate could not be rigorously ruled out (Otzen and Oliveberg, 2002Go). By profoundly destabilizing S6 with double mutations, it turns out that S6 can be ‘drawn out’ into providing more clear-cut evidence for an unfolding intermediate. Whereas the refolding plots remain linear, all of the double mutants show curvature in the unfolding limb, but the degree of curvature varies significantly (Figures 2 and 3). In particular, two mutants stand out, namely LA30/LA75 and IA8/LA79. For LA30/LA75, the unfolding limb of the chevron plot reaches a plateau around 5 M GdmCl and then declines slightly (Figure 2A). Even more remarkably, the mutant IA8/LA79 initially reaches a plateau in the unfolding limb around 2 M GdmCl and then starts to rise again above 5 M GdmCl (Figure 3A).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 2. Chevron plots for the mutants analysed in this study [wild-type and the single mutants LA30 and LA75 from Otzen et al. (1999)Go are included for comparison]. Data for wild-type analysed according to Scheme 1 and Equation 2 and data for the mutants according to Scheme 2 and Equation 3. For clarity, the same value range is used on the different y-axes. (A) (filled circles) S6 wild-type, (open squares) LA30, (filled squares) LA75, (x) LA30/LA75; (B) (filled circles) VA6/LA90, (open squares) LA26/LA79, (filled squares) AG35/MA67, (x) IA8/LA26; (C) (filled circles) EQ22/VA85, (open squares) VA6/LA30, (filled squares) LA30/VA37, (x) LA30/VA65; (D) (filled circles) VA37/VA65, (open squares) VA6/LA75, (filled squares) VA77/LA75, (x) IA8/VA88, (+) IA8/LA75.

 


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 3. (A) Chevron plot for IA8/LA79 fitted to Scheme 3 and Equation 4. The fit gives the following data: ; mf = 0.58 ± 0.14; ; mu = –0.02 ± 0.02; ; mI = 1.51 ± 0.10; logku* = 1.59 ± 0.05; mu* = –1.74 ± 0.12. (B) obtained from Scheme 2 (fitted to the entire chevron plot) versus log KD-Nwater obtained from Scheme 1 and Equation 2 (fitted to the linear section of the chevron plot). The line shows the best linear fit, which has a slope of 0.99 ± 0.04 and an intercept of 0.10 ± 0.10.

 
The purpose of this study was to compare the stabilities of the native state, transition state for unfolding and the collapsed state. However, the diversity in the unfolding behaviour of the double mutants makes it necessary to consider how the kinetic data are best analysed to obtain these stabilities. As is often the case in kinetics, several options are available.

For all double mutants except IA8/LA79, the curvature can be incorporated into the analysis either in a three-state model according to Scheme 2 (see Materials and methods) or by including a second-order term in the otherwise linear dependence of logku on [GdmCl]. We have previously favoured the latter option, as there was no consistent trend in the values for KI obtained for different single mutants (Otzen and Oliveberg, 2002Go) or for the mutant LA30 at different temperatures (Otzen and Oliveberg, 2004Go). However, the kinetic behaviour of the unfolding limb of IA8/LA79 is obviously incompatible with a simple second-order polynomial fit and LA30/LA75's unfolding limb only fits poorly (data not shown). For all mutants except IA8/LA79, the data can be fitted very well to Scheme 3; for LA30/LA75 in particular the fit is considerably better than a polynomial fit. The fit provides three constants, namely the refolding rate kf, the unfolding rate ku and the constant KI (and also their denaturant dependences). The equilibrium constant KD-N can be calculated using the relationship KD-N = kf/(kuKI). Data are summarized in Table II.

For IA8/LA79, the upward curvature in the unfolding limb above 5 M GdmCl can be included by a simple extension of Scheme 2 to include a direct unfolding step from N to D (Scheme 3). This leads to a satisfactory fit (Figure 3A). Upward curvature in the unfolding limb (but without an unfolding intermediate to complicate the picture) has also been seen for the ß-sandwich protein TI I127 and has been rationalized by the existence of two parallel unfolding pathways from the native state, of which one dominates at low and the other at high denaturant concentrations (Wright et al., 2003Go). In this case, a comprehensive analysis based on a large number of mutants with upward curvature was able to delineate the overall structure of the transition state connected to each unfolding pathway. For S6, only IA8/LA79 shows this behaviour and the paucity of ‘useful mutants’ precludes a more detailed analysis of this putative secondary unfolding pathway. Note that IA8/LA79 and LA30/LA75 are the only mutants with mI values (the dependence of logKI on [GdmCl]) close to zero. When interpreted within the framework of Schemes 2 and 3, this suggests that I and N are essentially equally compact. That is, I is native-like in compaction, since N has not changed structure significantly compared with wild-type; see below. All other mutants have mu values around 0.2–0.4 and therefore unfold through an I state which is more expanded than N. Thus a native-like intermediate may open up for alternative unfolding routes, provided that the mutant is suitably destabilized. LA30/LA75 has a midpoint of denaturation slightly higher than IA8/LA79 (0.72 vs 0.63 M GdmCl) and it is possible that an additional mutation in this mutant would open up an alternative unfolding pathway.

Although the fits to the chevron plots with downward curvature are very satisfactory, the errors on logKI and mI are considerable and these errors are carried over to KD-N. A simpler approach to analyse the double mutants is to exclude the curved region of the unfolding limb, restricting ourselves to the V-shaped region of the chevron plot, which may then be analysed according to a two-state model (Scheme 1). In this case, KD-N = kf/ku. Data are summarized in Table III. Clearly, the data are determined with less error in this case, as the analysis is simpler. However, there is a complication: The slopes of the refolding limbs and the linear regions of the unfolding limb also vary significantly among the mutants. These curvatures make energy differences between S6 wild-type, single and double mutants highly sensitive to denaturant conditions. Therefore, to allow us to calculate physically meaningful coupling energies and compare values for different mutants, all the unfolding and refolding rates were extrapolated to water, using the linear regions of the chevron plots (Table III). Stabilities derived from unfolding and refolding rates in water have been found to be consistent with equilibrium data when unfolding rates are extrapolated from the first linear region of the chevron plot above the denaturation midpoint (Otzen and Oliveberg, 2002Go).

There is also an important corollary in our analysis: whether the kinetic data are analysed according to a two-state model (restricted to the linear regions of the chevron plot) or a three-state model (including the whole range of denaturant concentrations) or even (in the case of the mutant IA8/LA79) as a triangular model, the data all extrapolate to the same equilibrium constant for unfolding in water () (Figure 3B). This is because for all the models, is equal to the difference in the extrapolated zero-molar intercepts of the linear parts of the refolding and unfolding limbs. In the two-state system, these values are and , respectively; in the three-state system, the values are and (), respectively. Since the calculated refolding rates and stabilities of the mutants are independent of the model to which they are fitted, but are determined most accurately using the simple two-state model, the following analysis of interaction energies will be confined to data based on the linear extrapolations.

The rate constants for folding and unfolding in Table III were used to calculate the interaction energies in Table IV using Equations 6GoGo9. The interaction activation energies for folding and unfolding are also included in Table IV. It is straightforward to calculate the activation energy of refolding, since the refolding rate constant (when measured in the absence of sodium sulfate) represents the step from the denatured state to the transition state. However, the strong evidence for the accumulating of an intermediate, following Scheme 3, during unfolding of several of the S6 mutants (LA30/LA75 and IA8/LA79) means that it is not possible simply to assign the observed unfolding rate constants to the step between the native state and the transition state for unfolding. The extrapolated rate of unfolding in water in this case is actually the product of ku and KI; see the previous paragraph. On the other hand, the lack of curvature for S6 wild-type means that it is not possible to calculate the stability of the putative unfolding intermediate and therefore ku and KI for wild-type cannot be separated. This prevents us from calculating the interaction energies involving I. In addition, the errors associated with the determination of KI for the other mutants would make any such {Delta}{Delta}G calculations so error-prone as to be virtually useless. Therefore, an apparent activation energy of unfolding which formally is a sum of the contributions from ku and KI is calculated. This does not affect the analysis of the properties of the transition state, which are analysed reliably from refolding data.


View this table:
[in this window]
[in a new window]
 
Table IV. Interaction energiesa

 
C shows significant coupling energies between side-chains

Table III also includes data on the stability of the off-pathway state C, determined by analysing the variation of the refolding rate constant with [GdmCl] in the presence of 1 M Na2SO4. Like the activation energy of folding, the determination of the stability of C only requires the use of refolding rate constants. The calculations of logKC and associated {Delta}{Delta}GC values are therefore not affected by the manner in which the unfolding data are interpreted.

Twelve of the 15 residue pairs (all except IA8/LA26, AG35/MA67 and LA30/VA37) show significant coupling energies (numerically between 2 and 0.35 kcal/mol) in N, and all except AG35/MA67 show coupling energies in C. These values are within the range of interaction energies reported for other systems. Coupling energies for hydrophobic residues generally lie around 0.2–1.5 kcal/mol (Otzen et al., 1995Go), and aromatic-aromatic and charge-aromatic contacts lead to about 1 kcal/mol of stabilization (Serrano et al., 1991Go; Loewenthal et al., 1992Go). Buried salt bridges can show up to 5 kcal/mol of stabilization because of the large penalty of burying single charges in a low dielectric medium (Fersht et al., 1985Go). Surprisingly, the coupling energies are generally greater in magnitude in C than in N, suggesting a large degree of well-defined side-chain contacts in C. Formally, a value of {Delta}{Delta}Gint = 0 does not necessarily imply a lack of coupling—it can simply mean that there is no change in pairwise interactions as the protein goes from the reference state (D) to the second state (C, TS or N). However, for this analysis it is assumed that D is essentially unstructured and therefore does not contain any persistent couplings (see discussion in next section).

There is no consistency between the magnitude and sign of the coupling energies in N and C. Eight double mutants have large differences between and , whereas the remaining seven show reasonable similarity. The two groups of side-chain pairs do not define distinct spatial regions of S6. Initial protein engineering analysis, based on single mutants, tentatively identified different domains of S6 with different levels of structure in C; one ‘blue’ region had a high level of structure, whereas the ‘red’ region had essentially no structure (Otzen and Oliveberg, 1999Go). However, there is no clear distinction between coupling energies involving ‘blue-only’ and ‘red-only’ residues (Table IV). The only distinction is that all positive (antagonistic) interaction energies in C involve red/red residues (except VA6/VA90, which is a red/blue pair). Therefore, although the data support the proposition that side-chain interactions are formed to a large extent in C, it is difficult to draw more detailed structural conclusions. A potential source of uncertainty in the analysis is the effect of the mutations on the structural properties of C. This effect may be considerable if C consists of a broad ensemble of states. The effect is particularly large for the single mutant VA85, where C has become considerably more compact than for wild-type (Otzen, 2005Go). However, VA85 is exceptional in the degree to which C is stabilized. For the mutants analysed here, the mC values, which are a gross indication of the change in solvent exposure between D and C, vary less than the value of mD-N, which reflects on the difference between D and N (Table III); the standard errors on mC and mD-N for the 15 mutants are 0.05 and 0.08, respectively. Hence the mutations appear not to affect C to a larger extent than N.

It is noteworthy that in C, Val6 shows positive (i.e. antagonistic) coupling with Leu30, Leu75 and Leu90; Leu30 shows antagonistic coupling with Val37 and Val65 and the two latter side chains are also antagonistically coupled. In contrast, there is attractive coupling between residues 30 and 75, which extends to residues 8, 26 and 79. Hence in C there is a local ‘antagonistic region’ centred around Val6, which appears to be surrounded by regions of attractive energies. This differs from the situation in N and TS, where there is a very large antagonistic interaction between Leu30 and Leu75 (see below), which extends to residues 37 and 65.

The gatekeeper residues Glu22 and Val85 interact strongly in C, suggesting that they cooperate in opening up an alternative folding pathway (C->N) upon mutation. The rate of folding along this pathway (k2) is 6.9 ± 0.4 s–1 for EQ22/VA85, whereas it is 10.5 ± 0.6 and 7.4 ± 0.2 s–1 for EQ22 and VA85, respectively. Since k2 for wild-type cannot be measured, the interaction energies between Glu22 and Val85 in the transition state between C and N cannot be calculated. However, the fact that it is very close to VA85 suggests substantial interaction: the interaction energy calculated according to Equation 7 essentially reduces to the term and is so small that it cannot be measured directly, i.e. , so .

A large and positive (antagonistic) coupling energy between Leu30 and Leu75 may be related to conformational strain

A remarkable feature is the very pronounced positive coupling energy between Leu30 and Leu75 in N and TS, although not in C. Positive coupling energy can be interpreted as an antagonistic rather than a synergistic effect. This is a very unexpected phenomenon. Horovitz et al. observed a small but significant positive coupling energy (0.33 kcal/mol) between Asp8 and Asp12 in a triad of charged residues on the surface of barnase (Horovitz et al., 1990Go). In this case, the coupling energy was most likely due to repulsion between the two negatively charged residues. The coupling energy between LA30 and LA75 is not only much larger (), it also involves hydrophobic residues where the repulsion is less obvious. Let us examine possible reasons for this phenomenon.

  1. Denaturant effects. The analysis of severely destabilizing mutants could potentially be complicated by non-linear denaturant effects at low denaturant concentrations where a general stabilizing salt effect might compete with denaturation potency. However, this can be ruled out on two grounds. First, antagonism is retained when concentration units are converted to activity units (data not shown), which precisely take such non-linear effects into consideration (Parker et al., 1995Go). Second, artefacts arising from such effects should show up for all severely destabilized mutants (including the double mutant IA8/LA79, which has a midpoint of denaturation around 0.63 M GdmCl), rather than a subset of the mutants.
  2. Effects in the denatured state. The m values listed in Table III are a gross indication of the changes in solvent-exposure between ground states D or N and transition state associated with unfolding (mu) and refolding (mf) (Tanford, 1970Go). Hence their sum (mD-N) indicates the change in solvent-exposure between D and N. Clearly, mD-N increases substantially for several of the double mutants, particularly those involving double Leu/Ile->Ala mutations. Since our crystallographic analysis (see below) shows that N is essentially unperturbed by the mutations, an increase in mD-N suggests that D becomes more extended. This is difficult to explain. A priori it would be expected that the truncation of large hydrophobic groups, all other things being equal, would reduce the solvent-accessible surface of D and thus reduce mD-N. Another possible explanation is that the denatured state from which folding occurs (in the absence of salt) is a collapsed or ‘off-pathway’ state D' with some intramolecular interactions; for the mutants with high mD-N values, this state could be replaced by the completely unstructured state D*. Truncation of large hydrophobic residues is known to eliminate structure in the denatured state of staphylococcal nuclease, leading to an increase in mD-N (Flanagan et al., 1993Go). However, it seems unlikely that the denatured state of S6 wild-type and other single-point mutants should represent a relatively compact collapsed state, since it is able to become even more compact and form a collapsed state C (which is distinct from D) in the presence of salt. Furthermore, mutations that destabilize D* will lead to a relative stabilization of N, which is opposite to what is seen for LA30/LA75, which is more destabilized than expected from the sum of the destabilizations of the individual mutations. It is also unexpected that mD-N and positive coupling energies should be coupled only for the mutation LA30/LA75 and not for the other double Leu/Ile->Ala mutants.
    From the definitions of the m values, it follows that the ratio ß{ddagger} = –mf/mD-N is a measure of how compact the transition state is relative to the native state, i.e. its position on the reaction coordinate. For the mutants with large values of mD-N, mf increases in parallel, so that ß{ddagger} is unchanged (Table III). Hence the gross features of the transition state appear to be unchanged by the mutations. This could mean that the mutations affect the binding of GdmCl locally in all three states, without necessarily reflecting on the overall compactness of the various states. In conclusion, changes in the denatured state do not offer an obvious explanation for the antagonistic behaviour of the LA30/LA75 mutant pair.
  3. General hydrophobic effect. The positive coupling energy stays remarkably constant at 2.05 ± 0.08 kcal/mol between 10 and 50°C (Table V). Over this range, the equilibrium constant for unfolding changes by a factor of 20–30. The temperature-invariance of shows that the coupling energy is not due to a classical hydrophobic effect; the hydrophobic effect declines at low temperatures, because it is dominated by entropic contributions (Privalov and Gill, 1988Go).
  4. Structural changes in the mutant native states. Antagonism could arise from strain between hydrophobic side chains due to close packing. In this scenario, relieving such strain in each of the single mutants would at least partially offset the loss of stability due to reduced surface area burial and loss of van der Waals contacts; while this would also be the case for the double mutant, the term ({Delta}{Delta}G1 + {Delta}{Delta}G2) in Equation 1 contains two ‘strain-relief’ contributions as opposed to the single contribution in the term {Delta}{Delta}G1+2. Such strain would a priori be considered unlikely, since hydrophobic interiors are in general rather plastic and will adjust to accommodate new groups or fill cavities (Karpusas et al., 1989Go; Varadarajan et al., 1992Go). Hence structural responses to hydrophobic truncations tend to reduce the degree of destabilization (Eriksson et al., 1992Go).


View this table:
[in this window]
[in a new window]
 
Table V. Temperature dependence of interaction energies in LA30/LA75a

 
However, consider the possibility that structural rearrangements could lead to unfavourable side-chain interactions in LA30/LA75. Sequence analysis shows that positions 30 and 75 are generally occupied by Leu and that variation at one position is not coupled with variation at the other (data not shown). To put these speculations on a firmer basis, the structures of LA30/LA75 and LA75 were determined by X-ray crystallography [that of LA30 was determined previously (Otzen et al., 1999Go)Go]. Initially there are no structural clues indicating unusual structural rearrangements. There is very little structural relaxation around the cavities created by the mutations (Figure 4). The cavity in LA30 has a volume of 95 Å3 whereas that in LA30/LA75 has a volume of 240 Å3. There is no cavity in S6 wild-type. The B-factors are not drastically increased around the sites of mutation. The average value goes from 23.83 Å2 (wild-type) to 26.42 Å2 (LA30) and 28.47 Å2 (LA30/LA75). There is no evidence for tightly bound water molecules in the cavities, which are lined entirely with hydrophobic amino acids.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 4. Cavity formation in the mutations LA30, LA75 and LA30/LA75.

 
Nonetheless, the dihedral angles for helix 1 (Glu18–Asn32) do change to some extent; the Ramachandran plot is significantly more scattered for LA30/LA75 than for LA30 and S6 wild-type, although the angles are in all cases well within the allowed region (Figure 5). Hence it could be that the effects of the mutations are not confined to the region of mutation. Subtle effects on the native state are also provided by fluorescence lifetime measurements, which reveal a shift in the amplitudes of the fast and slow components in the double mutant LA30/LA75 compared with wild-type and other mutants (data not shown). The single Trp (residue 62) is facing away from residues 30 and 75 and is closer to residues mutated in double mutants where the lifetimes are not affected, so the effect on the lifetime behaviour is likely to be propagated through global shifts in the structure rather than local environmental changes.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5. Ramachandran plots for residues 18–32 (helix 1) in wild-type S6 and the three mutants LA30, LA75 and LA30/LA75. The ({phi},{psi}) angles become more scattered for LA30/LA75 than for LA30 and S6 wild-type, although they in all cases remain within the allowed region.

 
Could antagonistic interactions have a role in the folding process?

Given that the strong antagonistic interactions between Leu30 and Leu75 are formed already in the transition state (although they become even more pronounced in the native state), it could be tempting to speculate that they represent a ‘reverse gatekeeper’ effect, that is, residues which encourage the exploration of other parts of the energy landscape by destabilizing both the transition state and the native state. However, there would appear to be no direct evolutionary advantage to such an effect. There are several additional arguments against this proposal. First, antagonism occurs to a comparable extent in the C-state and the antagonistic ‘hotspots’ overlap partially (namely residues 37 and 65 and, to a smaller extent, residues 30 and 75, cf. Table IV). Second, such ‘functional antagonism’ would be reflected in a rate constant that was lower than that expected from a protein of this structure. Absolute folding rate constants can be predicted to a certain extent, given the correlation between the logarithm of the folding rate constant and the contact order, which is the average distance in sequence between interacting residues. The lower the contact order (corresponding to a preponderance of local rather than global contacts), the higher is the folding rate (Plaxco et al., 1998Go). This correlation is particularly good for proteins with similar topology. S6's mixed {alpha}/ß topology is shared by the proteins U1A, Ada2 h, acyl phosphatase, MerP and HPr (Chiti et al., 1999Go). However, S6 falls well outside the linear correlation between logkf and contact order in this group. S6's contact order value of 0.19 predicts it should fold with a rate constant of 7–8 s–1, which is more than 50 times slower than the actual value (404 s–1). Hence S6 folds significantly faster than expected from its topology, indicating that antagonistic interactions do not have a role of reducing folding rates. Rather, the hotspots seem to be hard-wired into the protein whatever the direction folding takes.

In the absence of a direct role in slowing down folding, what is the significance of these antagonistic interactions? They may simply be a result of what Onuchic and Wolynes term ‘residual energetic frustration’ (Onuchic and Wolynes, 2004Go). Therefore, even though native contacts are overall stabilizing, they may be heterogeneous in magnitude, with some making large contributions and others actually detracting from overall stability. It has been pointed out that funnel landscapes with non-additive forces show increased cooperativity in folding. While this makes the transition state less diffuse, more polarized and more compact, it also favours the formation of partially structured states (Onuchic and Wolynes, 2004Go) which are potential traps for misfolding. Hence antagonistic interactions may to some extent temper this cooperativity and in this way serve the evolutionary purpose of making it easier to reach the native state.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The author is grateful for support from the Danish Technical Research Council. Ole Kristensen and Rajiv Vaid Basiawmoit are thanked for help with the crystallographic analysis, Eduardo Melo for lifetime measurements and Tony Clarke for initial discussions. This paper has been approved by Board Member Fabrizio Chiti.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Carter,P.J., Winter,G., Wilkinson,A.J. and Fersht,A.R. (1984) Cell, 38, 835–840.[CrossRef][ISI][Medline]

Chiti,F., Taddei,N., White,P.M., Bucciantini,M., Magherini,F., Stefani,M. and Dobson,C.M. (1999) Nat. Struct. Biol., 6, 1005–1009.[CrossRef][ISI][Medline]

Dill,K. (1999) Protein Sci., 8, 1166–1180.[Abstract]

Eriksson,A.E., Baase,W.A., Zhang,X.J., Heinz,D.W., Blaber,M., Baldwin,E.P. and Matthews,B.W. (1992) Science, 255, 178–183.[ISI][Medline]

Fersht,A.R., Shi,J.P., Knill-Jones,J., Lowe,D.M., Wilkinson,A.J., Blow,D.M., Brick,P., Carter,P., Waye,M.M.Y. and Winter,G. (1985) Nature, 314, 235–238.[CrossRef][ISI][Medline]

Flanagan,J.M., Kataoka,M., Fujisawa,T. and Engelman,D.M. (1993) Biochemistry, 32, 10359–10370.[CrossRef][ISI][Medline]

Hidalgo,P. and MacKinnon,R. (1995) Science, 268, 307–310.[ISI][Medline]

Horovitz,A. (1996) Fold. Des., 1, R121–R126.[ISI][Medline]

Horovitz,A. and Fersht,A.R. (1990) J. Mol. Biol., 214, 613–617.[CrossRef][ISI][Medline]

Horovitz,A. and Fersht,A.R. (1992) J. Mol. Biol., 224, 733–740.[CrossRef][ISI][Medline]

Horovitz,A., Serrano,L., Avron,B., Bycroft,M. and Fersht,A.R. (1990) J. Mol. Biol., 216, 1031–1044.[ISI][Medline]

Horovitz,A., Serrano,L. and Fersht,A.R. (1991) J. Mol. Biol., 219, 5–9.[CrossRef][ISI][Medline]

Itzhaki,L.S., Otzen,D.E. and Fersht,A.R. (1995) J. Mol. Biol., 254, 260–288.[CrossRef][ISI][Medline]

Jackson,S.E. and Fersht,A.R. (1993) Biochemistry, 32, 13909–13916.[CrossRef][ISI][Medline]

Karpusas,M., Baase,W.A., Matsumura,M. and Matthews,B.W. (1989) Proc. Natl Acad. Sci. USA, 86, 8237–8241.[Abstract/Free Full Text]

Lindahl,M. et al. (1994) EMBO J., 13, 1249–1254.[Abstract]

Loewenthal,R., Sancho,J. and Fersht,A.R. (1992) J. Mol. Biol., 224, 759–770.[CrossRef][ISI][Medline]

Maxwell,K.L. et al. (2005) Protein Sci., 14, 602–616.[Abstract/Free Full Text]

Mildvan,A.S. (2004) Biochemistry, in press.

Onuchic,J.N. and Wolynes,P.G. (2004) Curr. Opin. Struct. Biol., 14, 70–75.[CrossRef][ISI][Medline]

Onuchic,J.N., Socci,N.D., Luthey-Schulten,Z. and Wolynes,P.G. (1996) Fold. Des., 1, 441–450.[ISI][Medline]

Otzen,D.E. (2005) Biochim. Biophys. Acta, 1750, 146–153.[ISI][Medline]

Otzen,D.E. and Fersht,A.R. (1999) Protein Eng., 12, 41–45.[CrossRef][ISI][Medline]

Otzen,D.E. and Oliveberg,M. (1999) Proc. Natl Acad. Sci. USA, 96, 11746–11751.[Abstract/Free Full Text]

Otzen,D.E. and Oliveberg,M. (2002) J. Mol. Biol., 317, 613–627.[CrossRef][ISI][Medline]

Otzen,D.E. and Oliveberg,M. (2004) Protein Sci., 13, 3253–3263.[Abstract/Free Full Text]

Otzen,D.E., Rheinnecker,M. and Fersht,A.R. (1995) Biochemistry, 34, 13051–13058.[CrossRef][ISI][Medline]

Otzen,D.E., Kristensen,O., Proctor,M. and Oliveberg,O. (1999) Biochemistry, 38, 6499–6511.[CrossRef][ISI][Medline]

Parker,M.J., Spencer,J. and Clarke,A.R. (1995) J. Mol. Biol., 253, 771–786.[CrossRef][ISI][Medline]

Parker,M.J., Sessions,R.B., Badcoe,I.G. and Clarke,A.R. (1996) Fold. Des., 1, 145–156.[ISI][Medline]

Plaxco,K.W., Simons,K.T. and Baker,D. (1998) J. Mol. Biol., 277, 985–994.[CrossRef][ISI][Medline]

Privalov,P.L. and Gill,S.J. (1988) Adv. Protein Chem., 39, 191–234.[ISI][Medline]

Quiram,P.A., Jones,J.J. and Sine,S.M. (1999) J. Biol. Chem., 274, 19517–19524.[Abstract/Free Full Text]

Schreiber,G. and Fersht,A.R. (1995) J. Mol. Biol., 248, 478–486.[CrossRef][ISI][Medline]

Serrano,L., Bycroft,M. and Fersht,A.R. (1991) J. Mol. Biol., 218, 465–475.[CrossRef][ISI][Medline]

Tanford,C. (1970) Adv. Protein Chem., 24, 1–95.[Medline]

Varadarajan,R., Connelly,P.R., Sturtevant,J.M. and Richards,F.M. (1992) Biochemistry, 31, 1421–1426.[CrossRef][ISI][Medline]

Wells,J.A. (1990) Biochemistry, 29, 8509–8517.[CrossRef][ISI][Medline]

Wright,C.F., Lindorff-Larsen,K., Randles,L.G. and Clarke,J. (2003) Nat. Struct. Biol., 10, 658–62.[CrossRef][ISI][Medline]

Received August 29, 2005; accepted September 1, 2005.

Edited by Fabrizio Chiti





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
18/11/547    most recent
gzi063v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Otzen, D.
PubMed
PubMed Citation
Articles by Otzen, D.