The Folding Process of Acylphosphatase from Escherichia coli is Remarkably Accelerated by the Presence of a Disulfide Bond

doi:10.1016/j.jmb.2008.04.051

J. Mol. Biol. (2008) 379, 1107–1118

Available online at www.sciencedirect.com

The Folding Process of Acylphosphatase from Escherichia coli is Remarkably Accelerated by the Presence of a Disulfide Bond Claudia Parrini 1 , Francesco Bemporad 1 , Alessio Baroncelli 1 , Stefano Gianni 2 , Carlo Travaglini-Allocatelli 3 , Jonathan E. Kohn 4 , Matteo Ramazzotti 1 , Fabrizio Chiti 1 and Niccolò Taddei 1 ⁎ 1

Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Viale Morgagni 50, 50134 Firenze, Italy 2

Istituto di Biologia e Patologia Molecolari, CNR c/o Dipartimento di Scienze Biochimiche, Università di Roma “La Sapienza,” Piazzale A. Moro 5, 00185 Rome, Italy 3

Dipartimento di Scienze Biochimiche “A. Rossi Fanelli,” Università di Roma “La Sapienza,” Piazzale A. Moro 5, 00185 Rome, Italy 4 Department of Bioengineering, University of California, Berkeley, CA 94720, USA

Received 20 December 2007; received in revised form 21 April 2008; accepted 23 April 2008 Available online 30 April 2008 Edited by F. Schmid

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wildtype protein folds according to a two-state model with a very fast rate constant (kFH2O = 72,600 s− 1), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms. © 2008 Elsevier Ltd. All rights reserved.

Keywords: protein folding; three-state folding; intermediate; thiol; reduced

Introduction *Corresponding author. E-mail address: [email protected]. Abbreviations used: AcP, acylphosphatase; AcPDro2, AcP from Drosophila melanogaster; ANS, 8-anilinonaphthalene sulfonic acid; CD, circular dichroism; ctAcP, common-type AcP; DLS, dynamic light scattering; EcoAcP, AcP from Escherichia coli; GdnHCl, guanidinium hydrochloride; HypF-N, N-terminal domain of HypF from Escherichia coli; mAcP, human muscle AcP; mutEcoAcP, EcoAcP mutational variant C5A/C49A; RCO, relative contact order; SsoAcP, AcP from Sulfolobus solfataricus.

The folding of a polypeptide chain into a stable native conformation is one part of a complex free energy landscape that contains not only the folded, functional conformation and its precursor unfolded and partially folded ensembles but also the misfolded, aggregation-prone states, oligomers, and mature amyloid-like fibrils.1 The mechanism by which a protein folds into its functional state is important per se as it helps decipher the protein folding code, but it is also an essential prerequisite for any study aimed at clarifying the “reverse side” of the

0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.

1108 coin—that is, protein aggregation and amyloid fibril formation.1 In the last 20 years, details on the mechanisms of folding have been accumulating for an increasing number of proteins. Among the different approaches used to face this issue, the study of proteins belonging to the same structural families with significant sequence identities has become of primary relevance in order to gain insight into the role played by such factors as hydrophobicity, secondary structure propensities, conformational stability, and topology in determining the mechanism and kinetics of folding.2–5 Chain topology plays a crucial role in the folding process.6 A theoretical study suggested that in addition to size and conformational stability, chain topology markedly influences the rate at which proteins fold.7 In particular, the relative contact order (RCO), which is the average sequence separation of a residue pair forming an interaction in the native state, is a relevant metric for predicting a protein's folding rate, with a statistically significant correlation existing between the rate constant for folding and the RCO.7 It has also been demonstrated that increasing the hydrophobicity at positions that are not involved in the folding nucleus accelerates the folding process.8,9 This behavior can be attributed to the ability of hydrophobic moieties to facilitate the collapse of the polypeptide chain and, consequently, to favor the entropic stabilization of the transitionstate ensemble.8,9 Consistent with these findings, a significant correlation has been found between folding rate and hydrophobic content in the acylphosphatase (AcP)-like structural family.10 It is generally accepted that disulfide bonds play an important role in maintaining structural integrity and protein stability11; disulfide bonds also seem to play an important role in protein folding, as observed in different proteins such as the fungal phytase,12 the antibody light chain,13 and hen lysozyme.14 The presence of a disulfide bridge in the core of the protein

Folding of E. coli Acylphosphatase

may restrict the folding pathway by bringing residues of the folding nucleus into proximity, thus facilitating folding to the native state. The folding of proteins belonging to the AcP-like structural family has been studied extensively in recent years.10,15–18 These proteins are good model systems for studies in vitro due to their small size (b 100 amino acid residues), their monomeric state, and the absence of cofactors and disulfide bonds. The three-dimensional structure is known at atomic resolution for some members of this family and appears to be characterized in all cases by a fivestranded antiparallel β-sheet facing two α-helices, with the hydrophobic core formed at the interface between the helices and the sheet.19–25 A new AcP from Escherichia coli (EcoAcP) was recently isolated.26 It is a small protein composed of 95 residues whose three-dimensional solution structure has been determined by NMR.27 EcoAcP displays an α/β structure with a topology similar to that found in other members of the family so far studied (Fig. 1). Moreover, alignment of AcP sequences reveals that EcoAcP contains the conserved sequence Val-Gln-Gly-Val-X-X-Arg, which forms the loop involved in substrate binding and corresponding, in this specific case, to residues 14–20.27 It also contains an asparagine residue at position 38 suggested to be involved in catalysis.29 Compared with other members of the same structural family, this protein shows an unprecedented feature, a disulfide bond formed by the cysteine residues at positions 5 and 49. Other members of the family, such as human muscle AcP (mAcP) and AcP from Drosophila melanogaster (AcPDro2), contain a single cysteine residue only, while the N-terminal domain of the E. coli HypF (HypF-N) contains three cysteine residues. However, in all these cases, the cysteine residues are solvent exposed and reduced with no disulfide bond formed. The two cysteine residues found in EcoAcP, Cys5 and Cys49, at least under

Fig. 1. Front (a) and back (b) views of EcoAcP (Protein Data Bank code 2GV1) as determined by 1H NMR spectroscopy.27 The disulfide bond between residues 5 and 49 is highlighted in green. Red and blue colors indicate β-strands and α-helices, respectively. The figure was generated using VMD 1.8.3 for Win32.28

Folding of E. coli Acylphosphatase

1109

nonreducing conditions in vitro, are arranged to form a disulfide bond that links the N-terminal region of the protein and the fourth loop27 (Fig. 1). Interestingly, a number of open reading frames coding for putative AcPs in bacterial genomes (mostly Enterobacteriaceae, Legionellales, and Vibrionaceae) have shown cysteine residues at positions corresponding to those observed in EcoAcP, possibly forming a disulfide bridge as a possible consequence of direct phylogenesis or convergent evolution for a common environmental pressure.26 This indicates that the presence of a disulfide bond is not a rare feature in this structural family, particularly in bacteria. A mutational variant in which the two cysteine residues were replaced by alanine was produced and purified (EcoAcP mutational variant C5A/C49A, mutEcoAcP) in order to investigate the role of the disulfide bridge on the folding mechanism and folding rate of EcoAcP. The folding mechanism was investigated for both the wild-type EcoAcP and the mutant EcoAcP, and the results have been compared with those obtained previously for different members of the same structural family. We will show that while EcoAcP folds very rapidly, over 3 orders of magnitude faster than expected on the basis of its RCO and hydrophobicity, the mutant folds much slower in accordance with theory based on its RCO and hydrophobicity. We also show that the RCO, recalculated for the wild-type protein taking into account the disulfide bond as providing an alternate path for determining the shortest sequence separation between contacting residues, is a good predictor of the folding rate of this protein. These data indicate that disulfide bridges are important determinants of protein folding rates whose contribution to defining chain topology must be considered in order to permit accurate predictions of the rate of protein folding.

Results Structure of EcoAcP and mutEcoAcP Far-UV circular dichroism (CD) spectra of both EcoAcP and mutEcoAcP were acquired in 5 mM acetate buffer, pH 5.5, at 25 °C, under experimental conditions in which the proteins adopt a native state. Both EcoAcP and mutEcoAcP show a typical α/β spectrum (Fig. 2a), with minor differences in the region 205–220 nm. This difference suggests that the two proteins might have a slightly different content in secondary structure, due to the presence of the disulfide bond and/or to differences in amino acid sequence. The near-UV CD spectra of EcoAcP and mutEcoAcP were acquired under native and nonnative experimental conditions. Under native conditions, both variants show a positive peak at 286 nm, a major shoulder at 294 nm, and additional shoulders in the region 270–280 nm, suggesting low mobility of tryptophan, tyrosine, and phenylalanine residues and a compact tertiary structure in the na-

Fig. 2. (a) Far-UV CD spectra, (b) near-UV CD spectra, and (c) size distributions of the native states of EcoAcP (circles) and mutEcoAcP (squares).

tive state (Fig. 2b). Nevertheless, no significant difference can be detected between the near-UV CD spectra of EcoAcP and mutEcoAcP, suggesting that the three-dimensional structure of the native state of EcoAcP is not significantly perturbed as a consequence of the removal of the disulfide bond. Following protein denaturation in the presence of a high concentration of guanidinium hydrochloride (GdnHCl), the spectra become flat and the peaks observed under native conditions disappear (data not shown), reflecting the loss of compactness and tertiary structure upon denaturation. The size distributions observed for EcoAcP and mutEcoAcP measured by dynamic light scattering (DLS) show the presence of a peak at 3.6 ± 0.6 nm (Fig. 2c). This value, which is consistent with the size

1110 of native EcoAcP as determined in solution using NMR spectroscopy,27 does not change following the double mutation, indicating that the presence of the disulfide bond does not alter the compactness of the protein. Moreover, there is no evidence of accumulation of oligomeric species that could arise from an increased propensity to aggregation that sometimes follows amino acid replacements. An increased propensity to aggregate has been previously observed in largely destabilized mutational variants of numerous proteins.30,31 Equilibrium unfolding EcoAcP requires high concentrations of GdnHCl to achieve complete denaturation. Nonionic chaotropic agents, such as urea, are not effective at any concentration.26 Figure 3 shows the GdnHCl-induced unfolding curves of EcoAcP and mutEcoAcP measured at equilibrium at pH 5.5 and 28 °C. Unfolding was monitored using both tryptophan intrinsic fluorescence and the CD signal at 228 nm. The results obtained with the two spectroscopic probes are comparable for each protein (Fig. 3). Unfolding at equilibrium appears to be a reversible and cooperative event for both variants, showing a single sharp transition in the 4–6 and 1–3 M ranges for EcoAcP and mutEcoAcP, respectively (Fig. 3). The main thermodynamic parameters of the unfolding process were inferred from each equilibrium curve assuming a two-state model and using the equation described by Santoro and Bolen for a procedure of best fitting.32 The data obtained for the same variant from the intrinsic fluorescence and CD measurements are in close agreement with one another (Table 1), indicating that the loss of secondary structure and the increased accessibility of fluorophores to the solvent are coupled events during the unfolding process. EcoAcP displays a much higher structural stability compared with mutEcoAcP,

Fig. 3. Equilibrium GdnHCl-unfolding curves normalized to the fraction of folded protein: unfolding of EcoAcP (circles) and that of mutEcoAcP (squares) were followed by intrinsic fluorescence (filled symbols) and far-UV CD (open symbols). The continuous lines represent the best fits of the data points to the equation edited by Santoro and Bolen.32 The resulting thermodynamic parameters are listed in Table 1.

Folding of E. coli Acylphosphatase Table 1. Thermodynamic parameters of unfolding determined from equilibrium experiments Protein variant ΔGH2O (kJ mol− 1) a

EcoAcP EcoAcPb mutEcoAcPa mutEcoAcPb a b

m (kJ mol− 1 M− 1)

Cm (M)

6.7 ± 0.7 6.9 ± 0.9 9.5 ± 1.0 10.9 ± 0.5

4.6 ± 0.2 4.6 ± 0.2 1.7 ± 0.2 1.6 ± 0.2

31.3 ± 3.1 32.0 ± 4.2 16.4 ± 1.7 17.2 ± 0.8

Determined by intrinsic fluorescence. Determined by CD.

suggesting that the disulfide bond plays an important role in the stabilization of the three-dimensional structure of the protein. Such data are in agreement with previously reported results in which single cysteine EcoAcP variants were studied.26 Folding and unfolding kinetics as a function of GdnHCl concentration The presence of two tryptophan residues deeply buried in the hydrophobic core of EcoAcP allows the folding and unfolding processes to be monitored by following the change of the intrinsic fluorescence emission. Folding and unfolding reactions were investigated at 28 °C and pH 5.5, using both a stopped-flow device and a continuous-flow device. In the case of EcoAcP, the unfolding process was examined by GdnHCl concentration jump experiments using a stopped-flow apparatus, at final denaturant concentrations ranging from 4.5 to 8 M (Fig. 4). The fluorescence traces obtained were all fitted to single-exponential functions, suggesting that the unfolding reaction is a monophasic process at all GdnHCl concentrations studied.

Fig. 4. Dependence of the folding and unfolding rate constants on GdnHCl concentration. The natural logarithms of the observed rate constants for EcoAcP (circles) and mutEcoAcP (squares) are plotted as a function of GdnHCl concentration. Diamonds correspond to the data obtained by the continuous-flow device. The continuous lines represent the best fit of the data to the equation edited by Jackson and Fersht.33 For mutEcoAcP, the fitting was performed using data points in the 0.36–4.10 M GdnHCl range. The thermodynamic parameters resulting from this kinetic analysis are summarized in Table 2.

1111

Folding of E. coli Acylphosphatase

The folding reaction, monitored by the stoppedflow device, was initiated by diluting GdnHCldenatured EcoAcP in refolding buffer and studied in the presence of final GdnHCl concentrations ranging from 3 to 5 M (Fig. 4). At all denaturant concentrations investigated, the resulting traces were fitted satisfactorily by single-exponential functions, indicative of one single phase in the refolding process. Moreover, the fluorescence emission at time zero, extrapolated from the single-exponential function, corresponded to the fluorescence emission of the unfolded protein resulting from measurements at high GdnHCl concentrations and a linear extrapolation to 3–4 M GdnHCl. Given the high refolding rate, it was not possible to investigate the process at final GdnHCl concentrations lower than 3 M using the stopped-flow apparatus, since the change of the signal was completed within the dead time of the instrument (ca 10 ms). For this reason, a continuousflow apparatus with a shorter dead time (50 μs) was used. This enabled us to study the refolding reaction of EcoAcP in the presence of final GdnHCl concentrations ranging from 1 to 2 M (Fig. 4). As for the data obtained at higher denaturant concentrations, all the resulting fluorescence traces were well fitted to a single-exponential function. It is therefore possible to conclude that under all the conditions used in this study, EcoAcP appears to refold very rapidly in a two-state manner. Figure 4 shows that data points relative to the refolding process are missing between the final concentrations of 2 and 3 M GdnHCl. At these denaturant concentrations, the refolding process is too fast to be followed with the stopped-flow apparatus and too slow for the continuous-flow apparatus. Moreover, the refolding rate at final GdnHCl concentrations below 1 M is too high even for the continuous-flow technique. A kinetic trace at a final GdnHCl concentration of 0.5 M was acquired (data not shown) in order to understand whether the refolding rate decreases at low denaturant concentrations due to the presence of a possible rollover in the chevron plot. Under these conditions, the fluorescence change was completed within the dead time of the instrument, with no evidence of a refolding rate lowered by the accumulation of partially folded states, suggesting that refolding is still a cooperative two-state process. The folding and unfolding rate constants of EcoAcP (kF and kU) are strongly dependent on the denaturant concentration (Fig. 4). The chevron plot of EcoAcP appears to be linear in the whole range of GdnHCl concentrations studied, suggesting again that a two-state model is adequate to describe the folding behavior of this protein.33 The data of lnkF

and lnkU versus GdnHCl were analyzed using the equation edited by Jackson and Fersht,34 allowing the rate constants for folding and unfolding in the absence of denaturant (kFH2O and kUH2O) and their dependencies on denaturant concentration (mF and mU values) to be determined (Table 2). The estimates of ΔGH2O, Cm (midpoint of unfolding), and m values obtained from the kinetic data are in good agreement with those obtained from equilibrium experiments (Tables 1 and 2), confirming that the folding mechanism of EcoAcP follows a two-state model. Table 3 shows the kF values for all AcP-like family members studied so far assuming that the folding process in each case follows a pure two-state model. The rate constant for folding of EcoAcP is the highest in this group of proteins, ca 3×105 times higher than that of the slowest folding protein so far studied, mAcP. The kinetics of folding and unfolding of mutEcoAcP were also studied at 28 °C and pH 5.5. The unfolding process was examined by GdnHCl concentration jump experiments at final denaturant concentrations ranging from 1.6 to 6.6 M (Fig. 4). The traces obtained were all fitted to single-exponential functions, indicating that, similarly to EcoAcP, the unfolding process appears to be of two states. The refolding reaction was initiated by diluting the GdnHCl-unfolded protein in refolding buffer and studied in the presence of final GdnHCl concentrations ranging from 0.16 to 1.96 M (Fig. 4). All the resulting traces were fitted satisfactorily by singleexponential functions, indicating the presence of a single phase. In the case of the disulfide-lacking variant, the dependencies of the rate constants of folding and unfolding on denaturant concentration are different when compared with EcoAcP. The unfolding rate constant shows a dependence on GdnHCl concentration that decreases at high denaturant concentration (Fig. 4). This behavior, shown by a downward curvature of the ascending branch of the chevron plot, can be attributed to a movement of the transition-state ensemble along the reaction coordinate during unfolding.35 At GdnHCl concentrations above 0.4 M, the refolding rate is linearly dependent on the denaturant concentration (Fig. 4). The lnkF and lnkU values at GdnHCl concentrations higher than 0.4 M and lower than 4.1 M, respectively, were analyzed as previously done for the wild-type protein. The resulting data are listed in Table 2. Interestingly, the kF value extrapolated in the absence of denaturant is over 1000 times lower than that of EcoAcP. The estimates of ΔGH2O, Cm, and m values obtained from the kinetic data are in agreement with the data obtained from equilibrium experiments

Table 2. Kinetic and thermodynamic parameters from kinetic measurements Protein variant EcoAcP mutEcoAcP

kH2O (s− 1) F

kH2O (s− 1) U

mF

mU

ΔGH2O (kJ mol− 1)

m (kJ mol− 1 M− 1)

Cm (M)

72,600 ± 12,800 48.9 ± 7.7

0.3 ± 0.2 0.1 ± 0.02

− 1.9 ± 0.1 − 2.5 ± 0.2

0.8 ± 0.1 1.4 ± 0.1

31.2 ± 2.1 15.3 ± 0.8

6.7 ± 0.1 9.9 ± 0.2

4.7 ± 0.4 1.5 ± 0.1

1112

Folding of E. coli Acylphosphatase

Table 3. Refolding rate constants for AcP-like structural family members Protein mAcP15 ctAcP16 HypF-N17,a SsoAcP8,a AcPDro218 EcoAcP mutEcoAcP

kH2O (s− 1) F 0.23 ± 0.04 2.3 ± 0.5 230 ± 50 273 ± 60 7.5 ± 0.3 72,600 ± 12,800 48.9 ± 7.7

a These proteins do not show a two-state folding; the indicated values are those extrapolated assuming a two-state kinetic.

(Tables 1 and 2). These results clearly indicate that the folding of mutEcoAcP is a simple two-state process in the range of GdnHCl concentrations considered for this analysis. Folding of mutEcoAcP followed by different spectroscopic probes The chevron plot of mutEcoAcP clearly deviates from linearity at GdnHCl concentrations lower than 0.4 M, where a downward curvature, generally referred to as “rollover,” is observed (Fig. 4). This phenomenon can be attributed to the existence of partially folded states under low-denaturant solvent conditions. 36 The fluorescence signal of fully unfolded mutEcoAcP at 0.30 M GdnHCl, determined by linear extrapolation from fluorescence measurements performed on the unfolded protein at high GdnHCl concentrations, was compared with the fluorescence value at time zero of the recorded trace (Fig. 5a) to assess the presence of early conformational changes occurring within the dead time of the stopped-flow experiment. As in the case of the refolding experiment performed at 0.96 M GdnHCl final concentration (Fig. 5a, inset), where the lnkF value is in the linearity range, no evident discrepancy was found between the two fluorescence values. This would suggest the presence of intermediate species whose intrinsic fluorescence is similar to that of the unfolded polypeptide chain. To verify further the possible presence of a partially folded state populated during folding of mutEcoAcP, we also monitored the recovery of the CD ellipticity and that of the fluorescence of 8-anilino-naphthalene sulfonic acid (ANS) during folding. The 228-nm ellipticities of the unfolded protein at 0.33 and 0.90 M GdnHCl final concentrations, extrapolated from measurements performed at high GdnHCl concentrations, were compared with the ellipticities at time zero resulting from the refolding traces recorded under the corresponding conditions (Fig. 5b). Despite the low signal-to-noise ratio of the CD trace, the ellipticity of the unfolded protein at 0.33 M GdnHCl appears to be much higher (less negative) than that observed at time zero, indicating the accumulation, at this denaturant concentration, of a partially folded species with a considerable degree of secondary structure formed.

Fig. 5. Representative refolding traces of mutEcoAcP. (a) Refolding trace obtained in the presence of 0.30 M GdnHCl (main panel) and 0.96 M GdnHCl (inset) following the intrinsic fluorescence emission. The continuous lines represent the best fit of the data points to a single-exponential function corrected with a straight line. The squares represent the intrinsic fluorescence values of the fully unfolded protein extrapolated to the final experimental conditions from values measured at high denaturant concentrations. (b) Refolding trace obtained in the presence of 0.33 M GdnHCl (main panel) and 0.90 M GdnHCl (inset) following the far-UV CD ellipticity at 228 nm. The squares represent the ellipticities of the fully unfolded protein extrapolated to the final experimental conditions from values measured at high GdnHCl concentrations. (c) Refolding trace obtained monitoring the change in ANS fluorescence in the presence of 0.25 M GdnHCl. The continuous line through the data points represents the best fit to a single-exponential function corrected with a straight line. The square corresponds to the fluorescence emission of ANS in the presence of the fully unfolded protein. The value 1.0 corresponds to the fluorescence of free ANS in solution. U is the unfolded state; I, the intermediate species; and N, the native state.

Folding of E. coli Acylphosphatase

By contrast, the experiment performed at 0.90 M GdnHCl concentration suggests that this species is no longer formed during refolding (Fig. 5b, inset). ANS is a fluorescent dye known to bind exposed hydrophobic regions of polypeptide chains; in particular, species displaying clusters of hydrophobic residues partially exposed to the solvent often bind this dye, causing an enhancement of its fluorescence.37,38 ANS fluorescence was monitored after dilution of the GdnHCl-unfolded protein into a refolding buffer containing 0.25 M GdnHCl (Fig. 5c). The result shows that ANS fluorescence decreases during the process starting from a signal ca 60% higher than that of free ANS. Interestingly, the ANS fluorescence in the presence of the unfolded protein extrapolated to 0.25 M denaturant concentration is significantly lower than the experimental value found at time zero and is comparable with the signal of free ANS in solution. This suggests that the refolding of this mutant at a low denaturant concentration proceeds via the formation of a partially folded state characterized by the presence of hydrophobic clusters exposed to the solvent that are buried before the final folded conformation is attained. The relatively high value of ANS fluorescence in the presence of the fully native protein compared with the signal recorded in the presence of the unfolded EcoAcP is consistent with the equilibrium ANS spectra (data not shown) and can be related to the presence of hydrophobic residues exposed to the solvent in the native β-sheet.27

Discussion The role of the disulfide bond on protein stability and folding The loss of the disulfide bond does not alter significantly the native structure of EcoAcP, as revealed by far-UV and near-UV CD (Fig. 2a and b); neither does it seem to affect the compactness of the native fold as EcoAcP and mutEcoAcP have similar hydrodynamic diameters (Fig. 2c). By contrast, the disulfide bond appears to have a marked effect in both protein conformational stability and folding rate. In our studies, the ΔGH2O value of EcoAcP is twice higher than that reported for the mutational variant. These data are in agreement with previously published results describing a mutational variant of EcoAcP in which only one of the two cysteine residues is substituted by an alanine residue.26 Except for a few cases,39 it has been generally reported that disulfide bonds are responsible for a stabilization of the overall three-dimensional structure of proteins of different sizes, regardless of their structural organization.40–47 It is clear that the native disulfide bond of EcoAcP also has a marked influence on the folding process of this protein. EcoAcP exhibits two-state refolding across the whole range of denaturant concentrations studied, with no early folding event within the dead

1113 time of the instrument (Fig. 4). In contrast, mutEcoAcP shows a rollover in the chevron plot. The presence of this downward curvature in a chevron plot can be due to a number of causes. In some cases, it has been shown that changes in structure and position of the transition state along the folding coordinate occur at low denaturant concentration.35 This led some authors to the idea that the energy maximum in the folding reaction is represented by a broad barrier, as well as that transition states can move along this barrier as denaturant concentration varies.48 Finally, rollover can arise because of the formation of stable on-pathway or off-pathway intermediates36,49 and because of the ionic strength effects when guanidinium salts are used.50,51 Our studies show that the rollover present in the mutEcoAcP chevron plot is caused by the presence of a partially folded state accumulating during folding at GdnHCl concentrations lower than 0.4 M (Fig. 4). If this behavior were due to the presence of guanidinium salts, the EcoAcP chevron plot should also display the same behavior because the two experiments were done in the same conditions. Moreover, from the analysis of intrinsic fluorescence, CD, and ANS binding affinity, it is possible to get information about the structure of the folding intermediate (Fig. 5). This species is characterized by a substantial content of secondary structure and the formation of a rudimental hydrophobic core, although tryptophan residues are somewhat solvent exposed. Similar properties have previously been shown also in partially folded states of other proteins.13,52 Our results suggest that the removal of the disulfide bond causes a large reduction, by over 3 orders of magnitude, of the folding rate and has, by contrast, only a small influence on the unfolding rate. Indeed, the unfolding rate extrapolated to the absence of denaturant is comparable in the two protein variants with and without the disulfide bond (Table 2). This behavior has also been observed previously for other protein systems, although the enhancements in the refolding rates were lower in magnitude.46,53,54 The increase in protein folding rate depends also on the position of a disulfide bond. The acceleration of the folding process can be detected if the disulfide bond is located in a protein region involved in the early stage of the folding process.55 EcoAcP appears to behave similarly to other protein systems, but the extent of the folding rate deceleration upon disulfide bond removal is the highest among those reported in the literature, with the folding rate of mutEcoAcP decelerated by ca 1500 times relative to EcoAcP (Table 2). Comparing the sequence of EcoAcP with the sequences of other AcPs (e.g., mAcP), it is reasonable to suppose that the disulfide bond is located in a region of critical importance for the folding process. mutEcoAcP shows a higher dependence of conformational stability on GdnHCl concentration (i.e., the m value) as compared with the wild-type protein; this difference can be attributed to an increase of both the mF and mU values (Table 2). The m value

1114 reflects the change in solvent-accessible surface area following the unfolding process.56 DLS measurements suggest that the compactness of the native states of the two protein variants is the same (Fig. 2c). The difference between the two extrapolated m values is therefore attributable to a difference of compactness of the denatured states. The denatured state of mutEcoAcP is less compact than that of EcoAcP, probably because the disulfide bond acts as a conformational restraint. The transition states of the two proteins also exhibit different degrees of exposure to the solvent. The fact that the mU value of mutEcoAcP is higher than that of EcoAcP suggests that a larger part of the polypeptide chain becomes exposed to the solvent when the transition state for the (un)folding reaction is formed. Hence, while the native states of the two protein variants are comparable, the presence of the disulfide bond renders the unfolded and transition states of the protein more compact. Comparison between members of the AcP-like structural family EcoAcP folds via an extremely fast two-state process, with no accumulation of partially folded species, although the elimination of the disulfide bridge leads to the accumulation of such a species. Two-state behavior has been observed for the folding of mAcP, common-type AcP (ctAcP), and AcPDro2, although the rates are in all cases much lower.15,16,18 In contrast, HypF-N and the AcP from Sulfolobus solfataricus (SsoAcP) have been shown to fold through the accumulation of a partially folded species.10,17 In previous studies, the influence of some specific parameters on the folding rate of proteins from the AcP-like family has been investigated.10,17 In such studies, only proteins devoid of disulfide bridges were considered (mAcP, ctAcP, HypF-N, SsoAcP, and AcPDro2). A significant correlation has been observed between the folding rate and the hydrophobic content of the sequence, when the rate constant for folding of each protein is extrapolated from a two-state model.10 Figure 6 shows that the two parameters are still correlated when mutEcoAcP is considered (r = 0.893 and p b 0.05). On the contrary, the correlation fails for EcoAcP because its folding rate goes well beyond the value expected, due to the presence of the disulfide bond that dramatically accelerates the process (Fig. 6, empty circle). The correlation between hydrophobicity and folding rate is therefore significant only for those proteins that lack intramolecular disulfide bonds. The hydrophobicity of the sequence is not the only factor influencing the refolding rate. Previous studies have shown a very significant correlation between folding rate and RCO, a measure of the topology complexity that reflects the relative importance of local and nonlocal contacts to a protein's native structure.7 Using an RCO value for EcoAcP calculated by taking into account the presence of the disulfide bond, the correlation is significant (r = 0.907

Folding of E. coli Acylphosphatase

Fig. 6. Correlation between the natural logarithm of the rate constants for folding and hydrophobicity of the sequence for six members of the AcP-like family. The reported values of folding rate constants are those extrapolated assuming a two-state folding model. The p value lower than 0.05 indicates that the correlation is statistically significant. Folding rate constants for mAcP, ctAcP, HypF-N, SsoAcP, and AcPDro2 are those published previously.10,15–18 The open circle refers to the data point of EcoAcP and is not considered in the procedure of best fitting.

and p b 0.05) (Fig. 7a). The RCO value calculated for EcoAcP without considering the disulfide bond suggests that this protein is an outlier from the expected correlation (Fig. 7a, empty circle). Indeed, the very high refolding rate of EcoAcP cannot be explained unless the S–S bond is considered in the calculation of RCO. The contribution of a single disulfide bond to the RCO value is very important because the bridge is a very strong link in the protein native state. The disulfide bond has a significant impact on RCO by providing an alternate path for calculating the shortest sequence separation between contacting residues (see Materials and Methods for details). Figure 7b shows the highly significant correlation between RCO and refolding rate when both EcoAcP and mutEcoAcP are considered (r = 0.907 and p b 0.01). In this analysis, the RCO values considered for EcoAcP and mutEcoAcP are obviously those with and without considering the disulfide bonds, respectively. Conclusions The investigation of the folding process of EcoAcP presented here and the subsequent comparative analysis with other homologous proteins support the idea that many aspects of the folding process are not conserved between evolutionarily related proteins. The search of the native state requires different times among the various proteins and causes partially folded states to accumulate only in some cases. The disulfide bond that characterizes EcoAcP brings two distant regions of the sequence closer to each other, lowering the conformational entropy of the unfolded protein and explaining the extremely high rate of its folding process.

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Folding of E. coli Acylphosphatase

Materials and Methods Materials ANS and GdnHCl were purchased from Sigma-Aldrich (St. Louis, MO, USA). The mutEcoAcP variant was obtained using a QuickChange site-directed mutagenesis kit from Stratagene (La Jolla, CA, USA). The presence of the desired mutations was assessed by DNA sequencing. Both the EcoAcP and mutEcoAcP were expressed and purified as described previously.26 Protein purity was tested by both SDS-PAGE and electrospray ionization mass spectrometry. An extinction coefficient (ε280) of 1.615 mL mg− 1 cm− 1 was used to determine protein concentration by UV absorption measurements. CD measurements

Fig. 7. Correlation between the natural logarithm of the rate constants for folding and the RCO for members of the AcP-like family. (a) The p value lower than 0.05 indicates that the correlation is statistically significant. The RCO value of EcoAcP reported in the figure was calculated by considering the disulfide bridge as a chain branch point. The empty circle, corresponding to the RCO value of EcoAcP without considering the presence of the disulfide bond, is not considered in the procedure of best fitting. (b) The p value lower than 0.01 indicates that the correlation is statistically highly significant. The RCO values of EcoAcP and mutEcoAcP reported in the figure were calculated by considering and without considering the disulfide bridge as providing an alternate path for determining the shortest sequence separation between contacting residues, respectively. In both panels, the reported values for the folding rate constants are those extrapolated assuming a two-state folding model. Folding rate constants for mAcP, ctAcP, HypF-N, SsoAcP, and AcPDro2 are those published previously.10,15–18

Our analysis also confirms that RCO and hydrophobicity appear to be important determinants of folding rates. More importantly, the analysis has allowed the presence of a disulfide bond to be identified as an additional determinant of the folding rate and its contribution to be determined in quantitative terms. We have shown that it is possible and indeed necessary to redetermine the RCO value of a protein with a disulfide bond to predict its folding rate, ultimately allowing the spectrum of proteins for which we can predict the folding rate to include those characterized by disulfide bonds.

The measurements were carried using a Jasco J-810 spectropolarimeter (Great Dunmow, Essex, UK) equipped with a thermostated cell holder attached to a Thermo Haake C25P water bath (Karlsruhe, Germany). Far-UV CD measurements of the native EcoAcP (both wild-type and mutational variants) were acquired in the range 200– 260 nm using a 1.0-mm path quartz cuvette at a temperature of 25 °C. The proteins (0.4 mg mL− 1) were in 5 mM acetate buffer, pH 5.5. Each spectrum was recorded as the average of many scans. Near-UV CD spectra of both proteins were collected in both native and nonnative conditions. Experimental conditions for native polypeptides were 50 mM acetate buffer, pH 5.5, at a 0.4 mg mL− 1 protein concentration and 25 °C. The native proteins were unfolded in a high-concentration GdnHCl solution for 1 h at 25 °C to allow the reaction equilibrium to be attained. Final conditions were 50 mM acetate buffer, pH 5.5, 5 M GdnHCl, 0.4 mg mL− 1 protein, and 25 °C. The cuvette path length was 1 cm. DLS measurements The measurements were performed using a Zetasizer Nano S DLS device from Malvern Instruments (Malvern, Worcestershire, UK) thermostated with a Peltier system. Low-volume 12.5×4.5-mm disposable cells were employed. Refractive index and viscosity of water were used for acetate buffers; the refractive index indicated by the instrument (1.45) for a generic protein was used for EcoAcP. The samples were prepared at a final protein concentration of 0.4 mg mL− 1 in 50 mM acetate buffer, pH 5.5. Before the measurements, the samples were centrifuged (18,000 rpm for 5 min) in order to eliminate any impurity and preexisting large aggregates. The size distribution by intensity was recorded at 25 °C. The reported data are the average of three consecutive measurements. Equilibrium unfolding The equilibrium unfolding of EcoAcP and that of mutEcoAcP were investigated by measuring the intrinsic fluorescence of 30 protein samples containing different concentrations of GdnHCl, ranging from 0 to 8 M, in 50 mM acetate buffer, pH 5.5, at 28 °C. All samples were preequilibrated for 1 h at 28 °C. The final protein concentration in each sample was 0.02 mg mL− 1. Measurements were made using a Perkin Elmer LS-55 spectrofluorimeter (Wellesley, MA, USA) equipped with a

1116 thermostated cell holder attached to a Haake F8-C35 water bath. The excitation wavelength was fixed at 280 nm, and the fluorescence emission scans were collected from 290 to 440 nm using a scan speed of 100 nm/min. The fluorescence spectra analyzed resulted from the mean of three independent scans on the same sample. The change of fluorescence (measured at the mean wavelength maximum) as a function of denaturant concentration was fitted to an equation accounting for a two-state transition, as described by Santoro and Bolen.32 The free energy difference between the unfolded and the native states in the absence of denaturant (ΔGH2O), the dependence of ΔG on denaturant concentration (m value), and the midpoint of unfolding (Cm) were obtained. CD spectra in the range 200–250 nm were acquired for 25 samples containing EcoAcP and mutEcoAcP at a protein concentration of 0.2 mg mL− 1. Each sample was preequilibrated for 1 h at 28 °C and contained different concentrations of GdnHCl, ranging from 0 to 8 M. The mean residue ellipticity at 228 nm was plotted versus the GdnHCl concentration, and the resulting data were fitted as described previously. Kinetic measurements Unfolding and refolding reactions were followed using a Bio-Logic SFM-3 stopped-flow apparatus (Claix, France) equipped with a fluorimeter working at an excitation wavelength of 280 nm, using a filter to collect fluorescence above 320 nm. All the experiments were performed at 28 °C, in buffered solutions containing 50 mM acetate, pH 5.5, at a final protein concentration of 0.02 mg mL− 1. The unfolding experiments were initiated by a 20-fold dilution of the native protein into solutions containing GdnHCl at a concentration in the range of 4.5–8 M for EcoAcP and at that in the range of 1.6–6.6 M for mutEcoAcP. Similarly, refolding reactions were initiated by a 20-fold dilution of the GdnHCl-unfolded protein into solutions containing the appropriate concentrations of GdnHCl. The final GdnHCl concentration was in the range of 3–5 M for EcoAcP and was in that of 0.165– 1.960 M for mutEcoAcP. The dead time of the instrument spanned from 8.49 to 10.44 ms depending on the flow rate. The very fast refolding reactions of the wild-type protein were observed using an in-house-built ultrarapid mixer of design and methodology similar to those published by Shastry et al.57 The flow cell was purchased from Hellma (Germany). The mixed sample was illuminated with an A1010B Mercury–Xenon lamp (PTI, UK) at 280 nm, using a Model 101 Monochromator (PTI). Fluorescence was recorded with a Micromax CCD camera (Princeton Instrument, USA), with a typical exposure time of 1–3 s and employing a 320-nm-cutoff emission glass filter. An original pneumatically driven loading syringe unit was designed. Data were recorded at ca 3 bars, leading to a linear velocity in the flow cell of about 16 m s− 1. Folding was initiated by an 11-fold dilution of the GdnHCl-unfolded protein into solutions containing the appropriate concentrations of GdnHCl. The unfolding and refolding traces were all fitted to single-exponential functions. The natural logarithms of the unfolding rate constant, kU, and the folding rate constant, kF, were plotted against GdnHCl concentrations, and the resulting plots were fitted to the equation described by Jackson and Fersht.33 Thermodynamic data were extrapolated as described by Jackson and Fersht.33 Folding of mutEcoAcP was also monitored by far-UV CD at 228 nm using a Bio-Logic SFM-20 stopped-flow device coupled to the Jasco J-810 CD detection system

Folding of E. coli Acylphosphatase described previously. The folding reaction was initiated by a 10-fold dilution of mutEcoAcP unfolded in 3.3 M GdnHCl into refolding buffer. Final conditions were 0.33 M GdnHCl, 50 mM acetate, pH 5.5, and 28 °C at a protein concentration of 0.153 mg mL− 1 mutEcoAcP. In another experiment, unfolded mutEcoAcP was 10-fold diluted into a buffer containing GdnHCl to reach a final denaturant concentration equal to 0.9 M. In both cases, the ellipticity at 228 nm was followed immediately after injection. The dead time of the experiment was 18.5 ms. The signal of the buffered solution with no protein was subtracted from the averaged trace. ANS fluorescence measurements Folding of mutEcoAcP was monitored using ANS fluorescence as a spectroscopic probe with a Bio-Logic SFM-3 stopped-flow module coupled to a fluorescence detecting system. The folding reaction was carried out by a 10-fold dilution of 0.4 mg mL− 1 mutEcoAcP, unfolded in 2.5 M GdnHCl, into a refolding buffer containing 111 μM ANS in 50 mM acetate, pH 5.5. Final conditions were 0.25 M GdnHCl, 100 μM ANS, 50 mM acetate, pH 5.5, and 25 °C at a protein concentration of 0.04 mg mL− 1. Immediately after injection, the emission of the dye above 475 nm was followed using an excitation wavelength of 370 nm. The dead time of the stopped-flow apparatus was 10.4 ms. The resulting ANS fluorescence trace was fitted to a singleexponential function corrected with a straight line. In a second set of experiments, the unfolded protein was diluted into denaturing buffers containing ANS. Final conditions were 0.04 mg mL− 1 EcoAcP, a GdnHCl concentration ranging from 3 to 7 M, 100 μM ANS, 50 mM acetate, pH 5.5, and 25 °C. The equilibrium ANS fluorescence was plotted versus denaturant concentration, and the fluorescence of the dye in the presence of unfolded protein was extrapolated linearly to 0.25 M GdnHCl. RCO calculation RCO was calculated as previously described7 with the modification that the disulfide bond present in EcoAcP was considered as providing an alternate path to the backbone for determining the shortest sequence separation between contacting residues. For example, in a decapeptide with terminal cysteine residues linked by a disulfide bond, the sequence separation between the N- and C-terminal residues is nine without taking into account the disulfide bond. Considering the disulfide bond, however, the shortest sequence distance lies through the linkage, resulting in a sequence separation of one between the same terminal residues.

Acknowledgements This work was supported by grants from the Italian MIUR (projects PRIN 2005 027330 and FIRB RBIN04PWNC) and the EMBO Young Investigator Program (EMBO YIP 2005).

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