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PEDS Advance Access published online on January 5, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzm077
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

The folding pathway of an engineered circularly permuted PDZ domain

Ylva Ivarsson, Carlo Travaglini-Allocatelli, Veronica Morea, Maurizio Brunori and Stefano Gianni1

Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, Sapienza - Università di Roma ‘ La Sapienza’, Piazzale A. Moro 5, 00185 Rome, Italy

1 To whom correspondence should be addressed. E-mail: stefano.gianni{at}uniroma1.it


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
To understand the role of sequence connectivity in the folding pathway of a multi-state protein, we have analysed the folding kinetics of an engineered circularly permuted PDZ domain. This variant has been designed with the specific aim of posing two of the strands participating in the stabilisation of an early folding nucleus as contiguous elements in the primary structure. Folding of the circularly permuted PDZ2 has been explored by a variety of different experimental approaches including stopped-flow and continuous-flow kinetics, as well as ligand-induced folding experiments. Data reveal that although circular permutation introduces a significant destabilisation of the native state, a folding intermediate is stabilised and accumulated prior folding. Furthermore, quantitative analysis of the observed kinetics indicates an acceleration of the early folding events by more than two orders of magnitude. The results support the importance of sequence connectivity both in the mechanism and the speed of protein folding.

Keywords: circular permutation/intermediates/kinetics/protein folding/transition state


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Native state topology has been proposed to be one of the main factors in controlling both speed (Munoz and Eaton, 1999Go; Baker, 2000Go; Fersht, 2000Go) and mechanism (Nishimura et al., 2000Go; Friel et al., 2003Go; Travaglini-Allocatelli et al., 2004Go) of protein folding. The topological proximity of N- and C-termini in many native proteins has recently allowed to challenge this notion by ingenious experiments on topological mutants such as circular permutants, where the native N- and C-termini are joined and the sequence cleaved in a different position (Viguera et al., 1996Go; Otzen and Fersht, 1998Go; Li and Shakhnovich, 2001Go; Lindberg et al., 2002Go; Miller et al., 2002Go). Circular permutation has proven to be an extremely interesting approach to study the kinetics and thermodynamics of the protein folding reactions. In fact, although the native structure generally remains the same, the protein stability and the kinetic folding mechanism may either be greatly affected (Viguera et al., 1996Go; Lindberg et al., 2002Go) or remain largely unchanged (Otzen and Fersht, 1998Go).

PDZ domains are small (80–100 amino acids) globular protein–protein interacting modules, with important roles in molecular recognition (Harris and Lim, 2001Go; Jemth and Gianni, 2007Go). The typical fold of the canonical PDZ domains involves six β-strands and two {alpha}-helices (Fig. 1A). PDZ domains are generally part of multi-domain proteins and exert their function by recognising small target motifs, exploiting a specific binding groove. The folding of murine PDZ2 from PTP-BL has been thoroughly investigated both by experimental and theoretical approaches. PDZ2 folds through a three-state mechanism involving two different transition states (namely a denatured like TS1 and a native-like TS2) with an obligatory high-energy on-pathway folding intermediate (Gianni et al., 2005aGo, 2005bGo; Ivarsson et al., 2007Go). Molecular dynamics simulations using {phi}-values as structural restrains (Vendruscolo et al., 2001Go) clarified that TS1 consists of a heterogeneous ensemble of quite expanded structures (Gianni et al., 2007Go); nevertheless, in all structures a specific cluster of sufficiently stable interactions between residues in β-strands 1, 4 and 6 can be identified (Fig. 1B). The formation of a β-sheet involving contacts between residues from the N- and C-termini can hence be seen as a key-event in PDZ2 folding. Here, we investigate the folding mechanism of an engineered circularly permuted PDZ2 (cpPDZ2), where the canonical N-terminal strand is shifted to the C-terminus position (Fig. 1A); thus two of the strands participating in the stabilisation of the early folding nucleus are contiguous elements in the primary structure. We studied cpPDZ2 folding using a variety of different experimental approaches including stopped-flow and continuous-flow kinetics as well as ligand-induced folding experiments. The results on cpPDZ2 suggest that although this circular permutation significantly destabilises the native state, a folding intermediate is stabilised and folding turns from single exponential to double exponential. Furthermore, a comparison with PDZ2 folding suggests that such mutation speeds up the early events of protein folding, as revealed by a lower energy barrier for TS1.


Figure 1
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  		Fig. 1. (A) Structure of natively folded PDZ2 (PDB ID: 1GM1). The three β-strands 1, 4 and 6 are coloured yellow, orange and blue, respectively. The difference in sequence connectivity of PDZ2 and cpPDZ2 is indicated in a schematic representation. (B) Hypothetical structure of the early transition state in the folding of PDZ2 obtained by molecular dynamic simulations using {phi}-values as restrains (Gianni et al., 2007Go). Residues in the regions corresponding to the β-strands 1, 4 and 6, coloured as in A, form an early folding nucleus in PDZ2 folding.

 

   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Design of circularly permuted PDZ2

An engineered circular permutant of PDZ2 (cpPDZ2) was designed by cleaving between D22 and G23 [numbering according the murine PDZ2 structure 1GM1 (Walma et al., 2002Go)], and linking residues G99 and G11 together. Residues D22 and G23 occur in the structurally variable, solvent accessible loop between the structurally conserved regions including β-strands 2 and 3. The introduction of a cleavage site within this loop is therefore not expected to significantly affect the PDZ domain structure. In the structure of the PDZ-like domain of the stress sensor protease DegS (PDB ID: 1TE0), a natural circular permutant of mouse PDZ2, residues D338 and D339 (structurally equivalent to G99 and G11 of mouse PDZ2, respectively) are linked together and the main-chain conformation of all structurally equivalent secondary structure elements between the two domains is conserved. This further suggests that the linkage of residues G99 and G11 of PDZ2 should not affect significantly the rest of the structure.

As previously described for other PDZ domains (Chi et al., 2007Go), a tryptophan residue was introduced to function as a fluorescent probe (Y43W). A synthetic gene encoding cpPDZ2 was purchased from GENEART (Germany).

Protein expression and purification

The gene was subcloned into the expression-vector pET28(c) and protein was expressed in BL21(DE3) cells (Invitrogen). After induction with 1 mM IPTG, the cells were grown for 24 h at 25°C. The resulting hexahis-tagged protein formed inclusion bodies and was resuspended at high [urea]. The protein was purified on an S-Sepahorose ion exchange column (Amersham Biosciences) and eluted with a gradient of 0–2 M sodium chloride in 50 mM sodium phosphate pH 6.3.

Ligand-binding experiments

Ligand-binding kinetics of cpPDZ2 were monitored by Förster resonance energy transfer (FRET) between the fluorescence donor Y43W and a dansyl-group covalently attached to the N-terminal of the target peptide D-EQVSAV, as previously described (Gianni et al., 2005bGo; Gianni et al., 2006Go). The experiments were performed at pH 7.2, 0.4 M sodium sulphate and 10°C with a protein concentration of 1.5 µM and varying the peptide concentration between 10 and 80 µM. The excitation wavelength was 280 nm and the fluorescence emission was measured using a 330 ± 20 nm band-pass glass filter.

Equilibrium unfolding

Thermal denaturation of cpPDZ2 in 50 mM sodium phosphate buffer pH 7.2, in absence or presence of stabilising 0.4 M sodium sulphate, was monitored by far-UV CD (202 nm) on a JASCO circular dichroism spectrophotometer using a 0.1 cm quartz cuvette (Hellma) and a protein concentration of 16 µM.

Urea-induced equilibrium denaturation of cpPDZ2 was measured by following the decrease in Trp emission at different wavelengths on increasing urea concentration at 25°C, pH 7.2 in 50 mM sodium phosphate buffer in presence of 0.4 M sodium sulphate at a constant protein concentration of 1.5 µM.

(Un)Folding kinetics

Kinetic (un)folding experiments were performed using a Pi-star stopped-flow apparatus (Applied Photopysics, Leatherhead, UK) in 50 mM sodium phosphate, pH 7.2, in absence (10°C) or presence (25°C) of 0.4 M sodium sulphate. The protein sample was excited at 280 nm and the folding reaction was followed by the change in fluorescence using 320 or 360 nm cut-off filters. An 11-fold dilution of denatured or native protein in appropriate buffer initiated refolding and unfolding.

Ligand-induced folding

Peptide-induced folding experiments were carried out at pH 7.2, 25°C and 0.4 M sodium sulfate by mixing unfolded cpPDZ2 (i.e. in the presence of 1.0–4.8 M urea) with different concentrations of the peptide D-EQVSAV. The excitation wavelength was 280 nm and the fluorescence emission was measured using a 330 ± 20 nm band-pass glass filter.

Continuous-flow experiments

Ultra-fast kinetic refolding experiments were carried out using the in-house built continuous-flow apparatus equipped with an ultra-fast mixing device (dead-time 50 µs) as described elsewhere (Jemth et al., 2004Go; Gianni et al., 2006Go). Folding was initiated by 11-fold dilution in solutions containing 0.44 M sodium sulphate and different urea concentrations. Final protein concentration was typically 15 µM. The folding reaction was monitored by exciting the Trp at 280 nm and following the increase in fluorescence signal using a 360 nm cut-off filter.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
An engineered circularly permuted PDZ2 was designed posing two of the three β-strands participating in the early folding nucleus (β1 and β6) as contiguous elements in the primary structure (Fig. 1A). We tested the permissibility of such circular permutation in PDZ2 by performing classic ligand-binding experiments under pseudo-first-order conditions (Fig. 2). A dansylated target peptide D-EQVSAV has previously been shown to be a valid ligand for wild-type PDZ2 (kon = 5.0 ± 0.3 µM–1 s–1 and koff = 45 ± 3 s–1, Gianni et al., 2006Go). The kon of cpPDZ2 with the same peptide was 1.9 ± 0.1 µM–1 s–1 and the koff 40 ± 4 s–1 and the KD 20 µM. Hence, the kon of cpPDZ2 is 2.6 times lower, whereas the koff remains essentially unchanged. However, it can be noted that the values of cpPDZ2 are comparable to those of other previously characterised conservative single point mutants (Gianni et al., 2006Go). Thus, despite the change in sequence connectivity, the circularly permuted PDZ2 variant appears to fold into a native like structure, at least as judged by its ligand-binding properties.


Figure 2
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  		Fig. 2. Observed kinetic rate constants for binding the dansylated peptide D-EQVSAV to the wild-type PDZ2 and to cpPDZ2 as functions of peptide concentration. Data were recorded at constant protein concentration of 0.6 µM by monitoring the increase in fluorescence of the dansyl group at pH 7, 50 mM phosphate buffer and 0.4 M sodium sulphate. The lines are the best fit to a linear function. As a result of the circular permutation, the kon is lowered from 5.0 ± 0.3 µM–1 s–1 in the wild-type PDZ2 to 1.9 ± 0.1 µM–1 s–1 in cpPDZ2, whereas the koff remains essentially unchanged (45 and 40 s–1 for the wild-type and circular permutant, respectively).

 
Thermal denaturation of cpPDZ2 at pH 7.2 in absence and in the presence of 0.4 M sodium sulphate was monitored by far-UV CD as reported in Fig. 3A. The midpoint of the thermal denaturation in absence of sodium sulphate is 303 ± 1 K (the Tm of PDZ2 under the same experimental conditions being 321 ± 1 K), with an on-set of the transition at ~290 K. Hence, cpPDZ2 is partially unfolded at pH 7.2 and 25°C. By addition of 0.4 M sodium sulphate, the midpoint of the transition is increased to 326 ± 1 K.


Figure 3
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  		Fig. 3. Equilibrium unfolding of cpPDZ2 by (A) temperature and (B) urea. Thermal denaturation was carried out at pH 7.2 in 50 mM sodium phosphate buffer with and without 0.4 M sodium sulphate; the transition was followed by CD spectroscopy at 202 nm. The urea-induced unfolding was carried out at 25°C in the same buffer in presence of 0.4 M sodium sulphate, and it was followed by fluorescence emission at multiple wavelengths (320–380 nm). The data were globally fitted to a two-state transition.

 
The urea-induced equilibrium denaturation of cpPDZ2 in presence of 0.4 M sodium sulphate is reported in Fig. 3B. Assuming a standard two-state model (Jackson and Fersht, 1991Go), denaturation transitions were fitted to the equation:

Formula

where {Delta}Gd represents the free energy of folding at a concentration D of denaturant, mD-N is the slope of the transition and D50 is the midpoint of the denaturation transition. The equation used takes into account the pre- and post-transition baselines (Santoro and Bolen, 1988Go). A global fit of different wavelengths between 320 and 380 nm yielded a denaturation midpoint of 2.5 ± 0.1 M [urea], displaying a mD-N value of 1.1 ± 0.05 kcal mol–1 M–1. The mD-N value of cpPDZ2 is similar to that of PDZ2 (1.2 ± 0.1 kcal mol–1 M–1; Gianni et al., 2005aGo). Thus, we would conclude that cpPDZ2 likely displays a low residual structure content in the denatured state.

The kinetics of the cpPDZ2 (un)folding was investigated at pH 7.2 in absence and presence of 0.4 M sodium sulphate at 10°C and 25°C, respectively. The semi-logarithmic plots of the folding/unfolding rate constants of cpPDZ2 versus denaturant concentrations (chevron plots) measured by stopped-flow are shown in Fig. 4, together with the chevron plots of wild-type PDZ2 (also in absence and presence of 0.4 M sodium sulphate) at 25°C. The chevron plot of the circularly permuted protein indicates a significant destabilisation. In fact, when the (un)folding kinetics was investigated in the absence of stabilising salt, only the unfolding rate constants of cpPDZ2 were measurable. On the other hand, in presence of 0.4 M sodium sulphate, the chevron plot of cpPDZ2 is clearly V-shaped, indicating a simple two-state system without detectable intermediates. The data set was therefore fitted to an equation for apparent two-state kinetics. The observed rate constant kobs is the sum of the folding and unfolding rates, kobs = kf + ku and it is dependent on urea concentration according to Eq. (1):

Formula

where kf0 and ku0 are the folding and unfolding rate constants in the absence of denaturant and mf and mu reflect the dependence of these rate constants on denaturant concentration and correlate with the change in accessible surface area between the two ground states and the transition state in between. In the case of a two-state transition, the sum of mf and mu should equal mtot obtained from urea-induced equilibrium denaturation (Jackson and Fersht, 1991Go). However, the sum of mf (0.54 ± 0.03 kcal mol–1 M–1) and mu (0.21 ± 0.01 kcal mol–1 M–1) from the two-state fit is 0.75, which significantly different from the mtot obtained from the equilibrium analysis (1.1 kcal mol–1). Such a discrepancy between m-values obtained from equilibrium analysis and from kinetics may indicate the existence of a folding intermediate (Teilum et al., 2002Go; Mayor et al., 2003Go), which may form faster than the stopped-flow time scale. The sub-ms events in cpPDZ2 folding were therefore monitored using an in-house built continuous-flow apparatus equipped with an ultra-fast mixing device, with a dead time of ~50 µs (Shastry and Roder, 1998Go; Capaldi et al., 2001Go; Jemth et al., 2004Go). Under all conditions explored, we observed refolding traces in the microsecond time range conforming to a single exponential time course. A representative refolding trace monitored by continuous-flow is reported in Fig. 5.


Figure 4
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  		Fig. 4. Semi-logarithmic plots of folding/unfolding kinetics as a function of urea concentration (chevron plots). The chevron plots of cpPDZ2 in the absence (10°C) and presence (25°C) of 0.4 M sodium sulphate, in comparison with the chevron plots of wild-type PDZ2 (25°C) are shown. In the case of cpPDZ2, only the unfolding limb of the chevron plot could be obtained in absence of stabilising sodium sulphate. Note the deviation from linearity in the chevron plot of PDZ2 in absence of sodium sulphate indicating a more complex folding reaction than a simple two-state transition. Both cpPDZ2 and PDZ2 have linear chevron plots in presence of 0.4 M sodium sulphate.

 

Figure 5
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  		Fig. 5. Complete chevron plot of cpPDZ2. Refolding rate constants obtained from the continuous-flow experiments (filled square) are plotted together with the refolding rate constants obtained from the ligand-induced refolding experiments (inverted triangle) and by conventional stopped-flow dilution experiments (triangle). Data were globally fitted to a three-state folding mechanism as described in the Results section. The inset shows a typical trace from the continuous-flow dilution experiments obtained by mixing the denatured protein with buffer, to reach a final urea concentration of 2.3 M. All experiments were carried out at pH 7.2, 25°C and 0.4 M sodium sulphate.

 
Owing to the low stability of cpPDZ2, we decided to strengthen the quantitative analysis of (un)folding kinetics by performing ligand-induced refolding experiments. In these experiments, a large excess of the ligand peptide is added to the denatured protein, thereby trapping newly folded protein by binding, draining the equilibrium towards the folded state. It has been shown that when and if (i) the ligand interacts only with the native protein and (ii) the binding reaction is much faster than the folding process, a determination of folding rates can be obtained independently of the unfolding even under strongly denaturing conditions (Sanz and Fersht, 1994Go). We measured the ligand-induced folding rate constant for cpPDZ2 at different urea concentrations by mixing the denatured protein with an excess of the dansylated peptide D-EQVSAV (the concentration used varied from ~600 µM to circa 2.5 mM at low and high [urea], respectively). All the observed binding traces followed single exponential behaviour. Data were analysed as previously described (Ivarsson et al., 2007Go) and the full folding limb ranging from 0 to 5 M urea could be monitored (Fig. 5). This method eliminates the need for long and questionable extrapolations in the curve fitting and, as exemplified here, may be particularly useful to study the (un)folding kinetics of marginally stable proteins, which may display a poorly defined folding arm in the chevron plot.

The urea dependence of the relaxation rate constants for the fast ({lambda}1) and slow ({lambda}2) phases together with the folding rate independently determined by ligand induced binding experiments ({lambda}3) are reported in Fig. 5. The combined data set was globally fitted to the roots of a quadratic equation describing a three-state folding mechanism.

Formula


Formula


Formula


Formula

where P = (k1+k–1+k2+k–2) and q = ((k1 + k–1 + k2 + k–2)2–4(k1k2)–4(k1k–2)–4(k–1k–2))1/2.

The resulting rate constants of formation and breakage of the intermediate are k1 = 6000 ± 500 s–1 and k–1 = 300 ± 100 s–1 and the formation and breakage of the natively folded protein have the rate constants k2 = 130 ± 8 s–1 and k–2 = 5.3 ± 0.1 s–1, returning a total mD–N value of 1.15 kcal mol–1 M–1. However, since the two phases do not appear to be kinetically coupled (i.e. they take place on different time scales at all urea concentrations), it is at this point not possible to discriminate between the on- and off-pathway schemes, contrary to what previously observed for other systems (Bai, 1999Go; Capaldi et al., 2001Go; Travaglini-Allocatelli et al., 2003Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The docking of the N- and C-terminal segments appears to be a key event in the folding of PDZ2. Docking of terminal elements as a first step in folding has been suggested for several small proteins (Krishna and Englander, 2005Go). This observation implies that proteins need to overcome a large entropic barrier at the early stages of folding. Several evolutionary advantages have been proposed for such a trajectory, such as the vastly reduced conformational space to be searched during folding and the higher stability of the natively folded protein (Christopher and Baldwin, 1996Go). In agreement with this proposal, the stability of cpPDZ2, in which the N-terminal strand has been moved to the C-terminus, is vastly reduced. However, it should be noticed that the stability of a protein critically depends on optimal packing of side chains in the core so that the loss of even a small number of van der Waals interactions can result in a major increase in the unfolding rate. In other words, the introduced loop and the new opening might determine a suboptimal packing of residues resulting in a loss of stability, although the ligand-binding kinetics of cpPDZ2 indicates the protein to be folded into a functional, wild-type-like native state.

The strategy of circular permutation has been applied to a number of small protein domains aiming at the investigation of the stability and folding mechanisms upon change of sequence connectivity (Viguera et al., 1996Go; Otzen and Fersht, 1998Go; Li and Shakhnovich, 2001Go; Lindberg et al., 2002Go; Miller et al., 2002Go). A specific emphasis has been put on the concept of contact order, according to which, in general terms, a protein with many local contacts should fold faster than a protein with more long-range contacts (Plaxco et al., 1998Go). Here, we analysed the folding pathway of an engineered circular permutant of a PDZ domain, where two segments of the previously identified folding nucleus are posed as contiguous elements in the primary structure. We have previously shown that PDZ2 folds via a complex folding mechanism involving two energy barriers (a denatured-like TS1 and a native-like TS2) and a high-energy on-pathway intermediate. A comparative study on five different PDZ domains showed that members of this protein family fold via a consensus mechanism where the transition state TS1 is rate limiting in water for all PDZ domain tested. Surprisingly, although our circular permutation destabilises the native state ({Delta}{Delta}Gwt-cp~2 kcal mol–1), the folding kinetics of cpPDZ2 reveal a remarkable stabilisation of the folding intermediate, which accumulates transiently during folding. Furthermore, examination of all four microscopic rate constants allowed us to conclude that cpPDZ2 displays a lower TS1 energy barrier, the microscopic rate constant kDI turning from 35 ± 2 s–1 (in the case of PDZ2) to 6000 ± 500 s–1 (in the case of cpPDZ2). Thus, in agreement with theoretical predictions (Baker, 2000Go; Fersht, 2000Go) and with direct measurements of loop formation in disordered polypeptides (Buscaglia et al., 2006Go), reducing the sequence separation between elements constituting an early folding nucleus produces a remarkable increase of the folding rate constants.

The kinetic folding pathway of cpPDZ2 raises the intriguing question of the nature of the folding intermediate. In fact, by assuming that the cpPDZ2 folding intermediate represents a productive species on path to the native state, as is the case in the wild-type PDZ2 folding, it may be difficult to reconcile the observed stabilisation of the intermediate with a destabilisation of the native state. Lindberg and Oliveberg (2007)Go recently proposed pathway malleability to be a consensus feature of protein folding. In simple terms, when and if the denatured chain may reach its native conformation by means of different independent units (foldons), circular permutation involving the cleavage of a folding nucleus present in a dominant foldon may result in a different dominant folding pathway. In line with this hypothesis, it is tempting to speculate that although circular permutation stabilises the early events of PDZ folding (as mirrored by stabilisation of both TS1 and the folding intermediate), the main folding trajectory may be re-routed to different folding pathways. Under such conditions, contrary to what has been described for PDZ2, the observed intermediate may act as an off-pathway kinetic trap, which competes with productive folding.

In summary, we have here outlined the effects of a circular permutation on the folding mechanism of PDZ2. The most intriguing effect is the stabilisation of a folding intermediate that may be either on- or off-pathway. Whether or not the observed folding intermediate represents a stabilised version of the on-pathway intermediate detected in the PDZ2 folding remains to be found by extensive {phi}-value analysis.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Y.I. is supported by a grant from the Wenner-Gren Foundations (Sweden). Work partly supported by grants from the Italian Ministero dell'Università e della Ricerca (2005027330_005) to C.T.A.; University of Rome, Sapienza, project Ateneo 2006 to M. B.


   Footnotes
 
Abbreviations: PDZ, post-synaptic density-95/discs large/zonula occludens-1; PDZ2, the second PDZ domain from murine protein tyrosine phosphatase. Back

Edited by William A. Eaton


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
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Received November 19, 2007; revised November 19, 2007; accepted November 19, 2007.


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