Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark E-mail: dao{at}bio.aau.dk
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Abstract |
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Keywords: chevron plots/crystallography/interaction energies/kinetics/mutants
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Introduction |
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![]() | (1) |
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, 1993; Otzen and Fersht, 1999
). 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, 1995
).
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.
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Materials and methods |
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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:
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![]() | (2) |
To include the whole unfolding limb for all mutants except IA8/LA79, data were analysed according to Scheme 2 with the following equation:
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For the mutant IA8/LA79, Scheme 3 was applied with the accompanying equation:
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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, 1999):
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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, 1990, 1992
):
Interactions in terms of activation energies of refolding:
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![]() | (7) |
![]() | (8) |
![]() | (9) |
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Results and discussion |
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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., 1994) (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, 1999
). 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.
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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., 1999), 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, 2002
); however, an unfolding intermediate could not be rigorously ruled out (Otzen and Oliveberg, 2002
). 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).
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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, 2002) or for the mutant LA30 at different temperatures (Otzen and Oliveberg, 2004
). 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., 2003). 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 0.20.4 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, 2002).
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 69. 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
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.
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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 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 0.21.5 kcal/mol (Otzen et al., 1995), and aromatic-aromatic and charge-aromatic contacts lead to about 1 kcal/mol of stabilization (Serrano et al., 1991
; Loewenthal et al., 1992
). 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., 1985
). 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
Gint = 0 does not necessarily imply a lack of couplingit 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, 1999
). 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, 2005
). 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 (CN) upon mutation. The rate of folding along this pathway (k2) is 6.9 ± 0.4 s1 for EQ22/VA85, whereas it is 10.5 ± 0.6 and 7.4 ± 0.2 s1 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., 1990). 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.
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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., 1998). This correlation is particularly good for proteins with similar topology. S6's mixed
/ß topology is shared by the proteins U1A, Ada2 h, acyl phosphatase, MerP and HPr (Chiti et al., 1999
). 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 78 s1, which is more than 50 times slower than the actual value (404 s1). 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, 2004). 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, 2004
) 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.
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Acknowledgements |
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References |
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Chiti,F., Taddei,N., White,P.M., Bucciantini,M., Magherini,F., Stefani,M. and Dobson,C.M. (1999) Nat. Struct. Biol., 6, 10051009.[CrossRef][ISI][Medline]
Dill,K. (1999) Protein Sci., 8, 11661180.[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, 178183.[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, 235238.[CrossRef][ISI][Medline]
Flanagan,J.M., Kataoka,M., Fujisawa,T. and Engelman,D.M. (1993) Biochemistry, 32, 1035910370.[CrossRef][ISI][Medline]
Hidalgo,P. and MacKinnon,R. (1995) Science, 268, 307310.[ISI][Medline]
Horovitz,A. (1996) Fold. Des., 1, R121R126.[ISI][Medline]
Horovitz,A. and Fersht,A.R. (1990) J. Mol. Biol., 214, 613617.[CrossRef][ISI][Medline]
Horovitz,A. and Fersht,A.R. (1992) J. Mol. Biol., 224, 733740.[CrossRef][ISI][Medline]
Horovitz,A., Serrano,L., Avron,B., Bycroft,M. and Fersht,A.R. (1990) J. Mol. Biol., 216, 10311044.[ISI][Medline]
Horovitz,A., Serrano,L. and Fersht,A.R. (1991) J. Mol. Biol., 219, 59.[CrossRef][ISI][Medline]
Itzhaki,L.S., Otzen,D.E. and Fersht,A.R. (1995) J. Mol. Biol., 254, 260288.[CrossRef][ISI][Medline]
Jackson,S.E. and Fersht,A.R. (1993) Biochemistry, 32, 1390913916.[CrossRef][ISI][Medline]
Karpusas,M., Baase,W.A., Matsumura,M. and Matthews,B.W. (1989) Proc. Natl Acad. Sci. USA, 86, 82378241.
Lindahl,M. et al. (1994) EMBO J., 13, 12491254.[Abstract]
Loewenthal,R., Sancho,J. and Fersht,A.R. (1992) J. Mol. Biol., 224, 759770.[CrossRef][ISI][Medline]
Maxwell,K.L. et al. (2005) Protein Sci., 14, 602616.
Mildvan,A.S. (2004) Biochemistry, in press.
Onuchic,J.N. and Wolynes,P.G. (2004) Curr. Opin. Struct. Biol., 14, 7075.[CrossRef][ISI][Medline]
Onuchic,J.N., Socci,N.D., Luthey-Schulten,Z. and Wolynes,P.G. (1996) Fold. Des., 1, 441450.[ISI][Medline]
Otzen,D.E. (2005) Biochim. Biophys. Acta, 1750, 146153.[ISI][Medline]
Otzen,D.E. and Fersht,A.R. (1999) Protein Eng., 12, 4145.[CrossRef][ISI][Medline]
Otzen,D.E. and Oliveberg,M. (1999) Proc. Natl Acad. Sci. USA, 96, 1174611751.
Otzen,D.E. and Oliveberg,M. (2002) J. Mol. Biol., 317, 613627.[CrossRef][ISI][Medline]
Otzen,D.E. and Oliveberg,M. (2004) Protein Sci., 13, 32533263.
Otzen,D.E., Rheinnecker,M. and Fersht,A.R. (1995) Biochemistry, 34, 1305113058.[CrossRef][ISI][Medline]
Otzen,D.E., Kristensen,O., Proctor,M. and Oliveberg,O. (1999) Biochemistry, 38, 64996511.[CrossRef][ISI][Medline]
Parker,M.J., Spencer,J. and Clarke,A.R. (1995) J. Mol. Biol., 253, 771786.[CrossRef][ISI][Medline]
Parker,M.J., Sessions,R.B., Badcoe,I.G. and Clarke,A.R. (1996) Fold. Des., 1, 145156.[ISI][Medline]
Plaxco,K.W., Simons,K.T. and Baker,D. (1998) J. Mol. Biol., 277, 985994.[CrossRef][ISI][Medline]
Privalov,P.L. and Gill,S.J. (1988) Adv. Protein Chem., 39, 191234.[ISI][Medline]
Quiram,P.A., Jones,J.J. and Sine,S.M. (1999) J. Biol. Chem., 274, 1951719524.
Schreiber,G. and Fersht,A.R. (1995) J. Mol. Biol., 248, 478486.[CrossRef][ISI][Medline]
Serrano,L., Bycroft,M. and Fersht,A.R. (1991) J. Mol. Biol., 218, 465475.[CrossRef][ISI][Medline]
Tanford,C. (1970) Adv. Protein Chem., 24, 195.[Medline]
Varadarajan,R., Connelly,P.R., Sturtevant,J.M. and Richards,F.M. (1992) Biochemistry, 31, 14211426.[CrossRef][ISI][Medline]
Wells,J.A. (1990) Biochemistry, 29, 85098517.[CrossRef][ISI][Medline]
Wright,C.F., Lindorff-Larsen,K., Randles,L.G. and Clarke,J. (2003) Nat. Struct. Biol., 10, 65862.[CrossRef][ISI][Medline]
Received August 29, 2005; accepted September 1, 2005.
Edited by Fabrizio Chiti
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