Helix stability and hydrophobicity in the folding mechanism of the bacterial immunity protein Im9

Susanne Cranz-Mileva1,2, Claire T. Friel1 and Sheena E. Radford1,3

1School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

3 To whom correspondence should be addressed. E-mail: s.e.radford{at}leeds.ac.uk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recent models suggest that the mechanism of protein folding is determined by the balance between the stability of secondary structural elements and the hydrophobicity of the sequence. Here we determine the role of these factors in the folding kinetics of Im9* by altering the secondary structure propensity or hydrophobicity of helices I, II or IV by the substitution of residues at solvent exposed sites. The folding kinetics of each variant were measured at pH 7.0 and 10°C, under which conditions wild-type Im9* folds with two-state kinetics. We show that increasing the helicity of these sequences in regions known to be structured in the folding intermediate of Im7*, switches the folding of Im9* from a two- to three-state mechanism. By contrast, increasing the hydrophobicity of helices I or IV has no effect on the kinetic folding mechanism. Interestingly, however, increasing the hydrophobicity of solvent-exposed residues in helix II stabilizes the folding intermediate and the rate-limiting transition state, consistent with the view that this helix makes significant non-native interactions during folding. The results highlight the generic importance of intermediates in folding and show that such species can be populated by increasing helical propensity or by stabilizing inter-helix contacts through non-native interactions.

Keywords: folding/helical propensity/hydrophobicity/immunity proteins/intermediate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The role of {alpha}-helical propensity and hydrophobicity in determining the rate and mechanism of protein folding has been explored by a number of theoretical and experimental studies (Villegas et al., 1996Go; Lopez-Hernandez et al., 1997Go; Viguera et al., 1997Go, 2002Go; Poso et al., 2000Go; Beck et al., 2001Go; Islam et al., 2002Go; Chowdhury et al., 2003Go; Gong et al., 2003Go; Ivankov and Finkelstein, 2004Go; Meisner and Sosnick, 2004Go). These studies have shown that the rate of folding of proteins that fold with two-state-kinetics is dependent upon their content of secondary structure and its stability. For example, {alpha}-helical proteins formed from sequences with high local helical propensity have been suggested to fold by a diffusion–collision mechanism (Myers and Oas, 2001Go; Gianni et al., 2003Go), which describes a hierarchical process in which marginally stable elements of secondary structure collide and dock, forming intermediates with increasing stability and ultimately the native state (Karplus and Weaver, 1994Go). In other models, hydrophobic collapse dominates the early stages of folding, causing compaction of the protein and thereby defining geometric constraints in which the conformational search for the native state can take place (Baldwin, 1989Go). A third model, the nucleation–condensation model, proposes that secondary and tertiary structure are stabilized concomitantly with most, if not all, residues contributing towards the stability of the folding nucleus which characterizes the rate-limiting transition state (Otzen et al., 1994Go; Itzhaki et al., 1995Go). Which model dominates folding for a particular sequence, therefore, may be determined by the balance between the intrinsic stability of secondary structural elements and the propensity of the polypeptide chain to undergo hydrophobic collapse (Daggett and Fersht, 2003Go). Folding thus involves a continuum of models in which the balance between the content and stability of secondary structure and the hydrophobicity of a protein determines which mechanism dominates the folding reaction.

A number of studies on the role of helix stability in protein folding has shown that increasing the intrinsic stability of helices by substitution of solvent-exposed residues stabilizes the native protein (Munoz et al., 1996Go; Viguera et al., 1997Go; Taddei et al., 2000Go). In such cases, stabilization of the helices increases the rate of folding if the helix concerned is formed in the rate-limiting transition state, but not in the unfolded state. Alternatively, if the helix concerned is formed only after the rate-limiting transition state has been traversed, increasing helical propensity has no effect on the folding rate constant, but results in a reduction in the rate of unfolding. Altering helix propensity in this manner, therefore, is a powerful method of elucidating the role of individual secondary structural elements in the mechanism of folding.

Although the importance of hydrophobic collapse in folding has been appreciated for decades (Dill, 1990Go), predicting the effect of altering the hydrophobicity on the mechanism of protein folding is complex. For example, decreasing the size or hydrophobicity of residues involved in the folding nucleus and in the core of the native state slows folding and decreases protein stability (Richards and Lim, 1993Go; Northey et al., 2002Go). By contrast, increasing the size of hydrophobic side chains within the core can selectively alter the stability of the native state relative to the transition state (Northey et al., 2002Go). Increasing the hydrophobicity of residues that are solvent exposed in the native state can also destabilize the native protein relative to the unfolded state by the so-called reverse hydrophobic effect, which implicates non-native hydrophobic clusters in the unfolded state as the origin of this effect (Pakula and Sauer, 1990Go; Munoz et al., 1994Go; Viguera et al., 2002Go). The role of hydrophobicity in the mechanism of folding, therefore, depends on the role and environment of individual residues at each stage of folding.

Here we investigate the role of helical propensity and hydrophobicity in the kinetic folding mechanism of the immunity protein, Im9* [a hexahistidine-tagged version of the wild-type protein (Gorski et al., 2001Go)]. Previous results have shown that this four-helix protein (Figure 1a and b) folds with a two-state mechanism at pH 7.0 and 10°C (Ferguson et al., 1999Go; Gorski et al., 2001Go; Friel et al., 2003Go), whereas at lower pH an intermediate becomes populated during folding (Gorski et al., 2001Go). Interestingly, the Im9* homologue, Im7*, which shares ~60% sequence homology with Im9* (Figure 1c), folds with three-state kinetics involving the population of a stable intermediate under both sets of conditions (Ferguson et al., 1999Go; Gorski et al., 2001Go). Using mutational analysis and hydrogen exchange experiments, we have shown that the intermediate formed during the folding of Im7* contains three of the four native helices (helices I, II and IV) packed around a specific hydrophobic core that is stabilized by both native and non-native interactions (Capaldi et al., 2002Go; Gorski et al., 2004Go). The rate-determining step in folding involves the disruption of the non-native interactions so as to allow helix III to dock on to the developing structure stabilizing the protein in the native conformation (Capaldi et al., 2002Go). Using rational redesign of the Im9* sequence based on knowledge of the conformational properties of the Im7* intermediate (Capaldi et al., 2002Go), we have recently shown that Im9* also folds via a three-helical intermediate involving helices I, II and IV and that increasing the hydrophobicity of only a single residue in helix II (V37L or E41V) or helix IV (Im9* V71I) is sufficient to stabilize this species (Friel et al., 2004Go). Combining these three mutations in the Im9* variant V37L/E41V/V71I switches the folding mechanism of Im9* from two- to three-state at pH 7.0 (Friel et al., 2004Go). Importantly, this intermediate, and also that populated during Im7* folding, is more fluorescent than both the native and unfolded states, suggesting that the single tryptophan residue in these proteins becomes buried in a non-native environment early during folding. Im7* and Im9* thus fold with similar structural mechanisms despite the differences in their kinetic folding mechanisms at pH 7.0.



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Fig. 1. Ribbon diagram of the structure of Im9 illustrating its four-helical fold and the positions of the residues altered in this study. The images were prepared using MOLSCRIPT (Kraulis, 1991Go) and Raster3D (Merrit and Bacon, 1997Go) using the coordinates from the solution structure of Im9 (1IMQ) (Osborne et al., 1996Go). (a) Side chains mutated to engineer helix propensity. Residues shown in green were changed to obtain the variant H1 (A13E, Q17A, T20A T21R); side chains shown in blue were changed in the variant H2 (V34A, T38A); and side chains shown in pink were changed to create the variant H4 (S65D, N69A, T70A, Q73R). (b) Side chains mutated to alter the hydrophobicity of different helices. The residues shown in green were changed in the variant H1P (A13I, Q17I); side chains shown in blue were changed in the variant H2P (K35L, E42L) and residues shown in pink were changed in the variant H4P (N69V, Q73V). (c) Alignment of the sequences of Im7 and Im9. The positions of the four helices in Im9 are shown by boxes (Osborne et al., 1996Go). Mutations introduced to engineer the helix propensity and hydrophobicity of the sequence of Im9 are indicated.

 
Im7 and Im9 bind tightly and specifically to different colicin DNase domains, inhibiting the activity of these endonucleases until they are secreted from the bacterial host (Kleanthous and Walker, 2001Go). The sequence differences in Im7 and Im9, which define the binding specificity of these proteins, are most prevalent in helices I and II (Figure 1c) and result in the two proteins having different helix propensity in these regions (Figure 2a). The sequences of Im7 and Im9 also have different hydrophobicity profiles (Figure 2b); the C-terminal region of helix I is more hydrophobic in Im9, whereas helix II is more hydrophobic in Im7. This pair of proteins therefore provides an ideal system with which to dissect the role of sequence, secondary structure propensity and hydrophobicity in determining the kinetic mechanism of immunity protein folding.



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Fig. 2. (a), (c) and (e) AGADIR (Lacroix et al., 1998Go) predictions of the fractional helix population of the sequences of different immunity proteins as a function of residue number. (b), (d) and (f) hydrophobicity of the sequences of different immunity proteins as a function of residue number obtained using the program ProtScale, the hydrophobicity scale of Kyte and Doolittle (Kyte and Doolittle, 1982Go) and a window size of 7. (a) and (b) Im7 (red) and Im9 (black); (c) and (d) Im9 (black) and the variants H1 (green), H2 (blue) and H4 (pink); (e) and (f) Im9 (black) and the variants H1P (green), H2P (blue) and HP4 (pink). In panels (a) and (b) a gap has been left between residues 27 and 28 since the sequence of Im9 is one residue shorter than that of Im7 (see Figure 1c).

 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutagenesis, protein expression and purification

Site-directed mutations in Im9* were made using the QuikChange site-directed mutagenesis kit (Stratagene). The resulting proteins were over-expressed and purified as previously described (Gorski et al., 2001Go). The residue numbers referred to in this paper are those of untagged Im9. Every mutant was sequenced to confirm that the gene contained only the desired substitutions. All proteins studied were confirmed to be within 1 Da of the expected mass using electrospray ionization mass spectrometry (ESI-MS) and ≥95% pure as determined by SDS–PAGE.

Data collection

All folding and unfolding experiments were performed at 10°C as described previously using an Applied Photophysics SX18.MV stopped flow fluorimeter (Ferguson et al., 1999Go). Briefly, for refolding experiments the denatured protein (50 µM) in 50 mM sodium phosphate buffer, pH7.0, containing 2 mM DTT, 1 mM EDTA and 8 M urea was refolded by 1:10 (v/v) dilution into buffer containing various concentrations of urea. Refolding time courses were well described by a double exponential function, in which the slower second phase comprising <10% of the total amplitude has been attributed to folding events limited by proline isomerization (Ferguson et al., 1999Go). In the mutant H4 this slower transition was not observed. For unfolding experiments folded protein (50 µM) in the same buffer without urea was diluted 1:10 (v/v) into buffers containing different concentrations of urea. In all cases unfolding was well described by a single exponential function.

Data analysis

The observed rate constants for each data set were fitted to either a two-state model (Scheme 1) or a three-state model involving an on-pathway intermediate (Scheme 2) as discussed previously (Capaldi et al., 2001Go, 2002Go; Friel et al., 2004Go). where U, I and N represent the denatured, intermediate and native states, respectively and kxy is the microscopic rate constant for the conversion of x to y. Previous analysis of the folding of Im9* as a function of pH has shown that a model involving the movement of a single transition state cannot describe the complete data set (Gorski et al., 2001Go). A three-state model in which the formation of an on-pathway intermediate is assumed to occur in a pre-equilibrium step (Baldwin, 1996Go), therefore, was used to fit all of the data for the Im9* variants that fold with apparent three-state kinetics in this study. The data for all variants presented also fit well to a model involving the rapid formation of an off-pathway intermediate. In this case the parameters determined result in stabilities for the native protein and the intermediate that are identical to those obtained using the on-pathway model.



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Scheme 1
 


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Scheme 2
 
The logarithm of the rate constants for folding and unfolding versus the concentration of urea were fitted to either Equation 1 for the two-state transition (Scheme 1) or Equation 2 for three-state transition (Scheme 2) using the non-linear regression function in Origin 7.0 (Originlab).

(1)

(2)
where kxy is the rate constant for the conversion of state x to state y and mxy is the denaturant dependence of the rate constant. In Equation 2, Kui is the equilibrium constant for the formation of the intermediate (I) and Mui is the denaturant dependence of {Delta}Gui.

In all cases an excellent fit was obtained. The errors on the resulting parameters, cited in Tables I and II, are the standard errors resulting from the fit. Because of the uncertainty in the uniqueness of the fitted parameters, especially when the intermediate is not very stable, values of {Delta}Gxy less than 1 kJ/mol and Mxy that vary by less than 10% were not considered significant (Friel et al., 2004Go).


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Table I. Folding and unfolding parameters for Im9*, the Im9* variants with increased helix propensity H1, H2 and H4 and the Im9* variants with increased hydrophobicity H1P, H2P and H4P. The data were acquired at pH 7.0 or 6.0 and 10°C and were fitted to a model describing either a two- or three-state transition involving the formation of an on-pathway intermediate (see Materials and methods)

 

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Table II. Folding and unfolding parameters for variants of H2P determined from the best fit of the folding/unfolding kinetics at pH 6.0 and 10°C to a model describing a three-state transition with an on-pathway intermediate (see Materials and methods)

 


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Fig. 3. Denaturant dependence of the folding and unfolding rate constants of wild-type Im9* and the variants H1, H2 and H4 at pH 7.0 and 10°C. The continuous lines show the best fit of the data to a model describing a three-state transition involving an on-pathway intermediate, except the data for wild-type Im9*, which were fitted to a model describing a two-state transition (see Materials and methods).

 


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Fig. 4. (a) Denaturant dependence of the folding and unfolding rate constants of wild-type Im9* and the variants H1P and H4P at pH 7.0 and 10°C. The continuous lines show the best fit of the data to model describing a two-state transition (see Materials and methods). (b) Denaturant dependence of the folding and unfolding rate constants of the variant H2P at pH 7.0 and pH 6.0 at 10°C. The continuous line shows the best fit of the data to a model describing a three-state transition (see Materials and methods). The folding and unfolding kinetics of the wild-type protein at pH 7.0 (dashed line) and the Im9* variant V37L/E41V/V71I (triangles) also at pH 7.0 and 10°C are included for comparison.

 
{Phi}TS and {Phi}I values were calculated according to Equations 3 and 4:

(3)

(4)
where {Delta}{Delta}Gui is the change in free energy of the intermediate, {Delta}{Delta}Gu–ts is the change in free energy of the rate-limiting transition state, {Delta}{Delta}Gun is the change in free energy of the native state and wt and mut correspond to the wild-type and mutant proteins, respectively. {Delta}{Delta}Gui and {Delta}{Delta}Gu–ts were calculated as described by Capaldi et al. (2002)Go. Errors quoted on {Phi} values are mathematically propagated from the error on the individual parameters determined from the fit (Table II).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tailoring helical propensity

To investigate the role of helix propensity in Im9* folding, three variants of Im9* were created. In each variant the helical propensity of helices I, II or IV was increased by substitution of two or more solvent-exposed residues, designed according to the algorithm AGADIR (Lacroix et al., 1998Go). Helix III is unstructured in the folding of Im7* and Im9* until after the rate-limiting transition state is traversed (Capaldi et al., 2002Go; Friel et al., 2003Go). The sequence of this helix, therefore, was not changed as part of this study. The Im9* variants containing helix-stabilizing substitutions in helices I, II or IV are referred to as H1, H2 and H4, respectively (Figure 1a and c). In all variants, changes in the overall hydrophobicity of the sequence were minimized (Figure 2c and d). The variant H1 was obtained by changing the solvent-exposed residues, Q17 and T20 to Ala (Figure 1a and c). In addition, two mutations were introduced that stabilize the helix through interactions with the helix dipole (A13E and T21R). In the resulting quadruple mutant, A13E, Q17A, T20A and T21R, the helical propensity of helix I is increased by ~15% (Figure 2c). Whilst all four residues altered in this variant are highly solvent exposed, T20 and T21 form side-chain-side-chain contacts with residues in the loop between helices I and II in native Im9. Accordingly, the substitution T21S has been shown to destabilize native Im9* ({Delta}{Delta}Gun {approx} 3.0 kJ/mol) (Friel et al., 2003Go). In the variant H2, the solvent-exposed residues V34 and T38 were changed to Ala (Figure 1a and c). These substitutions increase the helical propensity of helix II by ~25% (Figure 2c), again without changing hydrophobicity (Figure 2d). Increasing the helical propensity of helix IV was more difficult, since several solvent-exposed residues in helix IV are already occupied by residues that have a high helical propensity (Figure 1c). In addition, we preferred not to alter the N- or C-terminal capping positions since the N-cap is occupied by proline and may play a role in defining the limits of this helix and the C-terminal capping position is occupied by glycine, mutation of which should destabilize the unfolded state entropically. Also, the single tryptophan residue (W74) could not be altered since it provides the fluorescent probe for folding. To create the mutant, H4, therefore, four mutations were made (Figure 1a and c). N69 and T70 were replaced with Ala, whilst the substitutions S65D and Q73R were included to stabilize the helix by interactions with the helix dipole. The substitution T70A is almost entirely responsible for the observed increase in helical propensity of ~10% (Figure 2c). However, this residue is not entirely solvent exposed in native Im9, its ß-CH3 group forming a van der Waals contact with Leu52 in helix III. Changing threonine to alanine abolishes this interaction and hence is expected to destabilize the native protein. Because helix III is not structured during Im9* folding until after the rate-limiting transition state has been traversed, the mutation T70A effectively increases the helical propensity of helix IV without perturbing non-local tertiary contacts formed in states prior to the native state.

Effect of increased helix propensity on the folding kinetics of Im9*

To ensure that each variant had folded correctly to a native-like structure, each protein was examined using far-UV circular dichroism (CD) and fluorescence. All of the proteins had a helical content similar to that of wild-type Im9* as judged by far-UV CD (data not shown). In addition, the fluorescence emission of the single tryptophan in all of the proteins is highly quenched, demonstrating that Trp74 packs closely against His46 in the core of these variants, as is observed for wild-type Im9* (Wallis et al., 1992Go; Osborne et al., 1996Go). Together these data demonstrate that the secondary and tertiary structure of the variants is not changed substantially relative to wild-type Im9*.

The folding and unfolding kinetics of wild-type Im9* at pH7.0 and 10°C are shown in Figure 3. The data show that the logarithm of the folding and unfolding rate constants of Im9* depends linearly on the denaturant concentration over the entire range studied, demonstrating that the protein folds with two-state kinetics under these conditions, in accord with previous results (Ferguson et al., 1999Go; Gorski et al., 2001Go; Friel et al., 2003Go).

As shown in Figure 3, increasing the helical propensity of helices I, II or IV has a dramatic effect on the folding kinetics of H1, H2 and H4 relative to wild-type Im9* in that the folding of all three variants is switched from a two- to a three-state mechanism. In no case was a change in the initial signal observed, demonstrating that akin to the intermediate formed during the folding of wild-type Im9* at low pH (Gorski et al., 2001Go; Friel et al., 2004Go), the intermediate formed during the folding of these variants has a fluorescence signal similar to that of the denatured state. Importantly, control experiments in which the refolding kinetics were measured as a function of protein concentration (0.3–88 µM in 1.0 M urea) showed no dependence of the rate constant for folding on protein concentration, demonstrating that the population of an intermediate during the folding of these variants cannot be attributed to transient intermolecular association over this concentration range.

Previous studies involving global analysis of the pH dependence of the folding kinetics of Im9* have demonstrated that the folding mechanism of Im9* at low pH (5.0–6.5) is best described by an on-pathway three state model; whereas other kinetic schemes such as movement of the rate-determining transition state do not describe the data well (Gorski et al., 2001Go). The folding kinetics of H1, H2 and H4, therefore, were also fitted to an on-pathway, three-state model in which an intermediate is populated rapidly in the dead time of folding (see Materials and methods). The resulting parameters (Table I) show that increasing the helix propensity of helices I, II or IV stabilizes the folding intermediate of Im9*, such that this species is now populated during folding ({Delta}Gui > 3.5 kJ/mol). In addition, the rate-limiting transition state is also stabilized such that all variants fold more rapidly than the wild-type protein in conditions under which the intermediate is not populated (Figure 3). By contrast, the stability of the native state of H1 is not changed by the amino acid substitutions introduced, whereas H4 is slightly destabilized ({Delta}{Delta}Gun = +0.1 and +1.7 kJ/mol, respectively). These effects presumably result from the removal of stabilizing interactions involving T20/T21 or T70 (see above). Only H2 is stabilized significantly ({Delta}{Delta}Gun = –2.0 kJ/mol). The data also show that the compactness of the intermediate and native states (revealed by the values Mui and Mun, where Mui is the denaturant dependence of the {Delta}Gui and Mun is the denaturant dependence of {Delta}Gun) are not affected greatly by the mutations introduced. Using these parameters as a measure of the reaction coordinate, the data show that increasing the helical propensity of helices I, II or IV results in the population of an intermediate in Im9* folding that is ~90% compact, whereas the rate-limiting transition state is ~96% compact, both relative to the native state. Similar values (~90 and ~99%) were obtained previously for the intermediate and rate-limiting transition states formed during the folding of wild-type Im9* at low pH (Gorski et al., 2001Go) and for the Im9* variant V37L/E41V/V71I at pH 7.0 (90% and 94%, respectively) (Friel et al., 2004Go).

In summary, therefore, increasing the helical propensity of any one of the three long helices in Im9* results in a switch in the folding mechanism of Im9* at pH 7.0 from two- to three-state, by increasing the stability of the intermediate such that it becomes significantly populated during folding. The data accord with previous results that suggest that helices I, II and IV are formed in the rate-limiting transition state in Im9* folding (Friel et al., 2003Go) and also in both the rate-limiting transition state and the intermediate of the Im9* variant (V37L/E41V/V71I), which populates a folding intermediate at neutral pH (Friel et al., 2004Go).

Altering surface hydrophobicity

In a second series of experiments, the effect of increasing the hydrophobicity of solvent exposed residues on the folding kinetics of Im9* was examined. Again, three variants were created, in each of which the hydrophobicity of helices I, II or IV was increased by substitutions at solvent-exposed sites, to create the variants H1P, H2P and H4P, respectively (Figure 1b and c). In each case, hydrophobicity was increased without significantly influencing the helix propensity of the resulting sequence (Figure 2e and f). Hence the Im9* variants A13I, Q17I (H1P); K35L, E42L (H2P); and N69V, Q73V (H4P) were created (Figure 1b and c). In the case of the variant H1P, the substitution of Ala and Gln with Ile slightly decreases the helical propensity of this sequence according to the helix prediction algorithm AGADIR (Figure 2e). However, substitution of these positions with residues that increase hydrophobicity to a similar extent (e.g. Leu) significantly increases the helical propensity of the sequence. Therefore, Ile was chosen as the preferred substitution at both positions. In the case of H2P and H4P the substitutions chosen have no significant effect on the helical propensity of these sequences as predicted by AGADIR (Figure 2e). All of the residues substituted are solvent exposed and none make any significant long-range interactions in native Im9.

Effect of increased surface hydrophobicity on the folding kinetics of Im9*

The folding and unfolding kinetics of the Im9* variants H1P, H2P and H4P at pH7.0 are shown in Figure 4a and b. Despite having significantly increased hydrophobicity, H1P and H4P fold with two-state kinetics at pH 7.0, demonstrating that collapse of the polypeptide chain in the early stages of folding of these proteins is highly specific (Figure 4a). Fitting the data to a two-state transition (see Materials and methods) showed that these variants are both destabilized significantly relative to wild-type Im9* [{Delta}{Delta}Gun ~= +4.0 kJ/mol (Table I)]. H1P and H4P also fold more slowly than wild-type Im9* (~2- and ~5-fold, respectively), whereas the rate of unfolding is not affected significantly by the amino acid substitutions introduced. The presence of hydrophobic residues at positions which are highly solvent exposed in the native state has been shown to stabilize the unfolded state of several proteins and thereby cause a net destabilization of the native protein through the so-called reverse hydrophobic effect (Pakula and Sauer, 1990Go; Munoz et al., 1994Go). Such an effect could explain the properties of H1P and H4P. Despite this, the value of Mun for these variants is not changed significantly relative to wild-type Im9*, suggesting that the relative compactness of the unfolded and native states is not affected by the amino acid substitutions introduced as judged by this analysis (Table I). A similar result has previously been obtained for a number of variants of the {alpha}-spectrin SH3 domain (Viguera et al., 2002Go). Interestingly, and in contrast with the results presented here for H1P and H4P, increasing the hydrophobicity of solvent-exposed residues in this SH3 domain also resulted in an increase in the rate of folding and unfolding, suggesting that stabilizing non-native hydrophobic interactions are formed in the transition state. The inability of the substitutions in helices I and IV to stabilize a folding intermediate, combined with the observed destabilization of both the rate-limiting transition state and the native state in these variants (Figure 4a), suggests that residues in these helices that are solvent exposed in the native state remain solvent exposed throughout folding.

By contrast with H1P and H4P, the folding kinetics of H2P display clear non-linearity of the logarithm of the observed rate constant versus urea concentration, suggesting that H2P populates an intermediate in the dead time of folding at pH 7.0 (Figure 4b). At this pH the intermediate is only marginally stable [{Delta}Gui = –4.2 kJ/mol (Table I)], but akin to the intermediate formed during the folding of Im9*, this species is stabilized at pH 6.0 ({Delta}Gui = –6.6 kJ/mol), whereas the native state is destabilized at the more acidic pH ({Delta}{Delta}Gun = +3.8 kJ/mol) (Figure 4b and Table I). Importantly, no dependence of the folding kinetics of H2P were observed on the protein concentration, over the range 0.1–8.5 µM at pH 6.0 in 1 M urea (data not shown), ruling out aggregation as the cause of the switch in the kinetic folding mechanism observed. The data suggest, therefore, that increasing the hydrophobicity of K35, E42 or both results in the population of a transient intermediate in Im9* folding by increased stabilization of this state, presumably through the formation of non-native hydrophobic interactions.

Comparison of the folding kinetics of H2P with previously published data on a series of Im9* variants, designed specifically to stabilize an intermediate in the folding of this protein, allows the origin of the switch in the folding mechanism of H2P to be rationalized at the level of individual residues (Friel et al., 2004Go). These studies showed that increasing the hydrophobicity of residues in Im9* at sites known to be important in stabilizing the intermediate in Im7* folding through the formation of non-native interactions also stabilizes the intermediate formed transiently during the folding of Im9*. For example, substitution of E41 with Val in helix II stabilizes the Im9* folding intermediate by ~4 kJ/mol at pH 6.0 (Friel et al., 2004Go). By contrast, increasing the hydrophobicity of individual residues at other sites in the variants E31L, V34L or V68I has no significant effect on folding. Together, these data suggest that the switch in folding kinetics of H2P from a two- to a three-state mechanism may result from the increased hydrophobicity of residue 42, although a contribution from residue 35 cannot be ruled out. By contrast with position 41, which is only partially exposed in native Im9, E42 is highly exposed to solvent. Substitution of residue 41 with a hydrophobic side chain thus stabilizes the native state of E41V relative to the wild-type protein (Friel et al., 2004Go), whereas the effects of the substitutions in the variant H2P on the native state are small (Table I).

In order to characterize the intermediate populated during the folding of H2P in more detail, a number of mutations were introduced and the properties of the intermediate and rate-limiting transition states were determined using {phi} value analysis (Fersht et al., 1992Go). The mutations were chosen based on previous analyses of the folding of Im7*, Im9* and the Im9* variant V37L/E41V/V71I (Capaldi et al., 2002Go; Friel et al., 2003Go, 2004Go) to report on (i) whether helices I, II and IV are docked together at different stages of folding (using the mutations, L16A, V37A, I67V, each of which is buried in the core of native Im9) and (ii) whether the intermediate and rate-determining transition states contain or lack helix III (using the mutation I53V). The kinetics of folding of each H2P variant were then measured at pH 6.0 and 10°C since the intermediate in the folding of H2P is more stable at this pH (Figure 4b and Table I). The resulting data (Figure 5 and Table II) (see also Figure S1, available as Supplementary data at PEDS Online) show that the intermediate populated during the folding of H2P resembles that formed during the folding of Im7* and the Im9* variant V37L/E41V/V71I (Capaldi et al., 2002Go; Friel et al., 2004Go) in that these species contain helices I and IV but lack helix III. Interestingly, however, the {Phi}I value for V37A in helix II in H2P is much smaller than that for L38A, the equivalent residue in Im7*. More information will be required to interpret the significance of this result and to determine the structure of helix II during the folding of H2P.



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Fig. 5. Comparison of (a) {Phi}I values and (b) {Phi}TS values for Im7* (hatched) and H2P (black). The {Phi} values for Im7* were obtained at pH 7.0, 0.4 M Na2SO4 at 10°C (Capaldi et al., 2002Go). {Phi} values for H2P were obtained at pH 6.0, 0 M Na2SO4 at 10°C (Table II). Note that Im7 is one residue longer than Im9 by the insertion of a single residue in loop 1. Residues 37/38, 53/54 and 67/68 thus denote equivalent positions on the two proteins.

 
Another common feature of immunity protein folding is the development of significant non-native interactions that stabilize the intermediate formed early during folding, but play a more minor role in the stabilization of the rate-limiting transition state ensemble. The mutations V37A and I67V probe for such events. For example, in Im7* the mutation L38A yields a {Phi}I value of 0.43 that decreases to 0.03 in the rate-limiting transition state, whereas the mutation I68V results in values for {Phi}I and {Phi}TS of 1.14 and 0.86, respectively (Figure 5) (Capaldi et al., 2002Go). Truncation of the equivalent residues V37 and I67 in Im9* H2P by their mutation to Ala or Val, respectively, yields {Phi}I values that are similar in magnitude to {Phi}TS (Figure 5 and Table II). Reorganizational events in the partially folded state in this protein, therefore, are not apparent. These data are in accordance with the observation that H2P folds and unfolds with a similar rate constant to wild-type Im9* under conditions in which folding is two-state (Figure 4b). By contrast, the rate of folding of the Im9* variant V37l/E41V/V71I (which was designed in order to stabilize an intermediate in the folding of Im9* at neutral pH) is significantly faster than that of the wild-type protein when folding is two-state (Figure 4b). These data indicate that, in contrast to H2P, the non-native interactions, which specifically stabilize the intermediate in V37l/E41V/V71I, persist to a degree in the rate-limiting transition state (Friel et al., 2004Go). Further analysis involving mutations at many sites, however, will be needed to compare the properties of the intermediate and rate-limiting transition states formed during the folding of Im7*, Im9* V37L/E41V/V71I and Im9* H2P in more detail.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
{alpha}-Helical propensity and the mechanism of immunity protein folding

Although two-state mechanisms have been the focus of protein folding studies in recent years, it is now becoming clear that intermediates may form during the folding of even the simplest proteins (Capaldi et al., 2002Go; Teilum et al., 2002Go; Mayor et al., 2003Go; Sanchez and Kiefhaber, 2003Go; Feng et al., 2004Go; Kamagata et al., 2004Go). Whereas in some proteins intermediates are unstable such that folding appears two-state [as is often observed for proteins shorter than 100 residues in length (Jackson, 1998Go)], the intermediates transiently formed during folding nonetheless may play an important role in defining the path to the native state. Although stable intermediates may slow the rate of folding, population of such species provides an important opportunity to delineate models for folding at the structural level. The immunity proteins thus provide a powerful system with which to investigate the structural properties of intermediate states and to test the role(s) of such species in folding. An intermediate has recently been shown to form during Im9* folding since the folding kinetics of this protein can be switched from two- to three-state in a number of ways, including reducing the pH (Gorski et al., 2001Go), making chimeras with the homologue, Im2 (Ferguson et al., 2001Go), and also by specifically stabilizing the intermediate state by rational redesign based on knowledge of the structure of the intermediate populated during Im7* folding (Friel et al., 2004Go). These data indicate that the structural mechanism of immunity protein folding is conserved despite the striking differences in the kinetic folding mechanisms of Im7* and Im9* at neutral pH. Here we show that the intermediate in Im9* folding can also be stabilized by increasing the helix propensity of any of the three long helices formed early during folding, and also by the introduction of specific non-native interactions that stabilize the hydrophobic core in the intermediate state.

By contrast with the strategy of using rational redesign to stabilize the intermediate in Im9* folding relative to both the unfolded and native states, which resulted in a highly stabilized intermediate species [{Delta}Gui = –10.5 kJ/mol for the Im9* variant V34I/E41V/V71I at pH 7.0 (Friel et al., 2004Go)], increasing helix propensity via the amino acid substitutions introduced into H1, H2 and H4 results in only small increases in stability relative to those achieved with Im9* V37L/E41V/V71I [{Delta}Gui = 3.6–6.1 kJ/mol for H1, H2 and H4 at pH 7.0 (Table I)], demonstrating that specific redesign of the hydrophobic core is a more effective way of stabilizing the intermediate formed during folding (Figure 6a). Detailed interpretation of the results of the helix redesign in terms of the energetic stabilization of individual species, however, is complex since altering helix propensity can also stabilize helical structure in the unfolded state. Further work using synthetic peptides or variants unable to fold at neutral pH will be needed to quantify the extent to which this occurs for the variants created here. Nonetheless, the data presented demonstrate that increasing the local stability of individual helices in Im9* provides one route of populating an intermediate during folding and reveal that differences in helical propensity play a significant role in determining the different kinetic folding mechanisms of Im7* and Im9* at neutral pH.



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Fig. 6. (a) Summary of the stability of the intermediates populated during the folding of Im7* and Im9*. All {Delta}Gui values are measured at pH 7.0, 10°C in the absence of Na2SO4. Data for Im7 are for the untagged protein under these conditions (Ferguson et al., 1999Go). Data for Im9* V37L/E41V/V71I (SIm9*) are taken from Friel et al. (2004)Go. Note that under these conditions wild-type Im9* folds with two-state kinetics (i.e. {Delta}Gui < 0.7 kJ/mol). (b) Stability of the native state of the same proteins shown in (a) relative to wild-type Im9* ({Delta}{Delta}Gun) is plotted. Under these conditions {Delta}Gun for wild-type Im9* is –27.4 kJ/mol (Table I). For clarity, increasing stability is shown as positive in both (a) and (b).

 
Populating intermediates by the optimization of non-native hydrophobic interactions

One of the most striking, and initially unexpected, results of this study was that increasing the hydrophobicity of helix II in the variant H2P switches the kinetic folding mechanism of Im9* from two- to three-state at neutral pH. By contrast, increasing the hydrophobicity of solvent-exposed residues in helices I or IV destabilizes native Im9*, presumably by destabilizing the folded state relative to the unfolded state through the reverse hydrophobic effect (Pakula and Sauer, 1990Go). For these variants, therefore, folding remains two-state. These data confirm previous results, demonstrating that collapse of the polypeptide chain in the early stages of folding is a highly specific event (Capaldi et al., 2002Go; Friel et al., 2003Go). The switch in folding kinetics of the variant H2P from two- to three-state can be attributed to the mutation E42L mirroring the effect of the substitution E41V in Im9* V37L/E41V/V71I, either by decreasing unfavourable electrostatic interactions and/or by allowing helix II to optimize the burial of hydrophobic amino acids in the developing hydrophobic core of the intermediate species. These data reinforce the importance of non-native interactions involving residues in helix II in determining the kinetic folding mechanism of the immunity proteins. The results support a diffusion–collision model for immunity protein folding, in which intermediates are predicted to be stabilized both by increased helix propensity and by increasing stabilizing contacts between helices by optimization of either native or non-native contacts (Beck et al., 2001Go; Islam et al., 2002Go). Finally, the results suggest that the formation of non-native interactions may be an important and generic event in folding, especially for proteins that fold by hierarchical mechanisms.


    Notes
 
2 Present address: University of Zurich, Biochemical Institute, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Back


    Acknowledgments
 
We thank Keith Ainley for technical assistance and Alison Ashcroft for mass spectrometry. We acknowledge with thanks Godfrey Beddard, Graham Spence, Stuart Knowling and members of the Radford group for their helpful contributions throughout this work. C.T.F. is supported by the Biotechnology and Biological Sciences Research Council (BBSRC). S.E.R. is a BBSRC Professorial Fellow. S.C.-M. was supported by the award of a Marie Curie Host Fellowship for Early Stage Training. This work was supported by the BBSRC, the University of Leeds and the Wellcome Trust.


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 Results
 Discussion
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Received January 18, 2005; accepted January 24, 2005.





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