Báo cáo khoa học: Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein pot

Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein Sı ´ lvia Bronsoms, Josep Villanueva, Francesc Canals, Enrique Querol and Francesc X. Aviles Institut de Biotecnologia i Biomedicina and Departament de Bioquı ´ mica i Biologia Molecular, Universitat Auto ` noma de Barcelona, Spain Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue pro- tein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been pre- viously reported to affect folding of the PCI mature form. The extent to which the Ônative-likeÕ form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli.However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form. Keywords: pro-region; protein folding; structure; disulfide; protease inhibitor. Proteins contain within their amino acid sequence the required information for their folding. However, other factors may be required for a fast and efficient folding in vivo. Molecular chaperones facilitate folding by decreas- ing the tendency of partially folded proteins to go into non- productive pathways [1]. The protein disulfide isomerase and the peptidyl-prolyl-isomerase can also function as folding catalysts [2,3]. Apart from these components that have a broad substrate specificity, the folding process may be also affected specifically by the precursor protein. Many proteins are synthesized in vivo as precursors in the form of prepro-proteins. Pre- or signal peptides are often involved in sorting, while pro-peptides or pro-regions can regulate many processes [4]. Depending on their function they can be classified in two groups [5]: the class I pro-peptides, which are required for the correct folding of the proteins to which they are attached [6–8] and the class II pro-peptides, which influence other cellular processes, such as secretion, protein activity or molecular assembly [9]. Class I pro-peptides have also been termed as Ôintra- molecular chaperonesÕ, and their role in folding has been demonstrated both in vitro and in vivo [7]. There are not many examples of the role of the pro-regions in small disulfide-rich proteins. In these proteins, the folding process differs from that of larger proteins in that is strongly constrained by the formation of the disulfide bridges. Among the most studied proteins of this group we find the bovine pancreatic trypsin inhibitor (BPTI). Its N-terminal pro-region contains a cysteine residue which appears to increase both the yield of properly folded mature BPTI and therateofthefoldingprocessin vitro [10], providing an intramolecular thiol-disulfide catalyst. Nevertheless, it did not appear to have any positive effect under physiological conditions [11]. Similarly, the pro-region of the guanylyl cyclase activating peptide (GCAPII) contributes signifi- cantly to the correct disulfide-coupled folding of the mature protein and the dimerization of the molecule [12]. In contrast, the studies performed with x-conotoxins demon- strated that the mature forms of these molecules contain sufficient information to direct their folding and correct disulfide pairing in vitro [13,14]. In all cases, the in vitro folding of the mature disulfide-rich protein is characterized by a low efficiency and a slow kinetics. This fact suggests that these proteins need other factors, apart from their mature amino acid sequence, in order to fold efficiently and rapidly into the native form in vivo. The nature of these factors, whether they are found in their pro-region or in Correspondence to F. X. Aviles and J. Villanueva, Institut de Biotecnologia i Biomedicina, Universitat Auto ` noma de Barcelona, 08193 Bellaterra (Barcelona), Spain. Fax: + 34 93 5812011, Tel.: + 34 93 5811315, E-mail: fxaviles@einstein.uab.es and villanj1@mskcc.org Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; CPA, carb- oxypeptidase A; Cys-Cys, cystine; PCI, potato carboxypeptidase inhibitor; ProNtPCI, PCI with the N-terminal pro-sequence; ProPCI, PCI with the N- and C-terminal pro-sequences; ProNt + PCI, PCI with the N-terminal pro-sequence added in trans; RP-HPLC, reversed- phase high performance liquid chromatography. (Received 8 May 2003, revised 7 July 2003, accepted 16 July 2003) Eur. J. Biochem. 270, 3641–3650 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03754.x other cellular components (protein disulfide isomerases, molecular chaperones …), seems to depend on each individual protein. Potato carboxypeptidase inhibitor (PCI) is a 39-residue globular protein that inhibits several metallocarboxypep- tidases [15]. It has a 27-residue central core with three disulfide bridges that forms a T-knot scaffold, also found in other proteins such as many growth factors [16]. This molecule is synthesized as a prepro-protein that, besides the 39-residue mature protein, contains a long 27-residue N-terminal pro-region of unknown function and a seven- residue C-terminal pro-region, probably involved in trans- port to the vacuoles [17]. The folding and unfolding pathways of mature PCI have been previously studied by our group and are well characterized [18,19]. The extremely inefficient folding of PCI in vitro [18], together with the presence of the above-mentioned long pro-sequences, suggest a possible involvement of them in the in vivo folding of PCI. Here, using different protein variants, we have investi- gated the role of both pro-sequences in the in vitro refolding of PCI, together with studies of the influence of the N-terminal pro-region on its in vivo expression. Both studies conclude that the pro-regions do not significantly influence the folding of PCI. Experimental procedures Plasmid constructs and mutagenesis A plasmid containing a synthetic gene encoding for the major isoform of PCI (IIa)[20] cloned into pINIII-OmpA3 vector [21], was used as a template to generate the plasmidic constructions used for the expression of the PCI forms studied. ProNtPCI was obtained by means of one-step PCR, subcloned into pGEM-T Vector System (Promega), restricted with XbaIandEcoRI and ligated into pIN-III- OmpA3 vector. Similarly, this construction was used as a template to generate the D3, Y37G and G35P/P36G ProNtPCI mutant genes, by means of one-step PCR. Constructs for D3, Y37G and G35P/P36G PCI mutant genes were achieved by PCR of wild-type mature PCI [22]. ProNtPCI was also cloned into pBAT4 expression vector [23], derived from the pET plasmids [24], with and without the leader sequence OmpA. ProPCI was generated from ProNtPCI by means of one-step PCR and cloned into pBAT4 vector without the leader sequence OmpA. All constructs cloned into pINIII vector were transformed into Escherichia coli strain MC1061 and those cloned into pBAT4 vector were transformed into E. coli strain BL21(DE3). Protein expression and purification For constructs cloned into pINIIIOmpA3 vector, E. coli MC1061 cells were grown at 37 °C and expression was induced at 0.1 attenuation unit at 550 nm by the addition of 1m M isopropyl thio-b- D -galactoside, and they were har- vested by centrifugation (13 000 g at 4 °C for 45 min) 20 h after induction of protein expression. ProNtPCI was purified from the culture medium by a Sep-Pak C 18 (Waters) cartridge and eluted with 70% isopropanol containing 0.1% trifluoroacetic acid. The protein was finally purified by RP-HPLC, on a protein C4 0.46 · 25 cm, 5 lm column (Vydac). The conditions used were: solvent A was water containing 0.1% trifluoroacetic acid, solvent B was acetonitrile containing 0.1% trifluoro- acetic acid and the gradient was 25–55% solvent B in 60 min. Details regarding the PCI purification protocol have been published elsewhere [20]. ProNtPCI mutant proteins used in the in vivo refolding experiments were directly analyzed by RP-HPLC on a Nova-Pak C8 3.9 · 150 mm column (Waters), after sample acidification with trifluoroacetic acid and filtration through 4 mm, 0.2 lm poly(vinylidene diflu- oride) filters (National Scientific). For protein production in E. coli BL21(DE3) cells, the cultures were grown until they reached a value of 1 attenuation unit at 550nm, induced by addition of 0.2 m M isopropyl thio-b- D -galactoside, and cells were harvested by centrifugation 2.5 h after induction. The cell pellet from a 1 L culture was resuspended in 50 mL 20 m M Tris/HCl, 0.5 m M EDTA (pH 8.5) and was maintained on ice for 10 min. The solution was sonicated for 10 min on ice at 50 Hz at half power, on a Labsonic-Braun sonicator and centrifuged at 22 000 g for 25 min. The pellet was resus- pended in 50 mL 20 m M Tris/HCl, 0.5 m M EDTA and 2% Triton X-100 (pH 8.5) and centrifuged at 22 000 g for 25 min. The pellet was resuspended in 10 mL 6 M guani- dinium chloride and 30 m M dithiothreitol (pH 8.5). After 6 h the sample was centrifuged at 3000 g for 10 min and the supernatant was dialyzed against 0.1 M Tris/HCl (pH 8.5) for 12 h and then renaturation was performed by dialysis in the presence of a redox system containing 4m M Cys and 2 m M Cys-Cys (cystine) at pH 8.5 for 48 h at 4 °C with a 3500-Da cut-off membrane (Spectrum Medical Industries Inc). After dialysis, the sample was centrifuged at 3000 g for 10 min and the supernatant was purified by RP-HPLC, on a Protein C4 0.46 · 25 cm, 5 lm column (Vydac). The peptide corresponding to the N-terminal pro-sequence (ProNt) was obtained by solid- phase chemical synthesis. The released peptide was purified by RP-HPLC on a Protein C4 1 · 25 cm, 5 lm column (Vydac), in a linear gradient 20–27% in 7 min and 27–40% solvent B in 26 min. In vitro folding experiments One hundred micrograms of lyophilized aliquots of PCI, 185 lg lyophilized aliquots of ProPCI and 171 lg lyophi- lized aliquots of ProNtPCI were used in each folding experiment. The proteins were dissolved in 0.5 mL Tris/HCl (0.5 M , pH 8.5) containing 5 M guanidinium chloride and 30m M dithiothreitol, to a final protein concentration of 46.5 l M . After 2 h at 25 °C, the reduced and denatured proteins were passed through a PD-10 (Pharmacia) column equilibrated with 0.1 M Tris/HCl buffer (pH 8.5). The proteins were eluted in 1.2 mL and split in three parts which were diluted to a final protein concentration of 14.5 l M , with the 0.1 M Tris/HCl buffer (pH 8.5), the same buffer containing 1 m M Cys and the same buffer containing 4 m M Cys and 2 m M Cys-Cys, respectively. In the experiments where the N-terminal pro-sequence was tested in trans,the peptide was added to the denatured and reduced PCI in the dilution buffer, to a final concentration of 14.5 l M .Samples 3642 S. Bronsoms et al. (Eur. J. Biochem. 270) Ó FEBS 2003 of all reaction mixtures were collected in a time-course manner for up to 24 h and trapped by mixing with an equal volume of: (a) 1% trifluoroacetic acid in water (reversible trapping) followed by analysis by RP-HPLC on a Protein C4 0.46 · 25 cm, 5 lm column (Vydac). The gradient was linear: 20–40% solvent B in 30 min for PCI, 25–35% solvent B in 10 min and 35–45% solvent B in 40 min for ProNtPCI and 20–30% solvent B in 5 min and 30–50% solvent B in 30 min for ProPCI; (b) 0.1 M iodoacetic acid in Tris/HCl buffer (0.5 M , pH 6.5) containing 40% (by volume) of dimethylformamide (irreversible trapping) [25]. Carboxymethylation was allowed to proceed for 30 min at 25 °C. Inhibitory activity The substrate used to perform the carboxypeptidase activity was 0.2 m M furyl-acryloyl- L -phenylalanyl- L -phenyl- alanine and the buffer was 50 m M Tris/HCl, 0.5 M NaCl, pH 7.5. To 985 lL of substrate, 5 lL of bovine carboxypeptidase A (CPA) (Sigma) at 0.02 mgÆmL )1 were added and the absorbance change at 330 nm was measured during 2 min; then 10 lLofincreasing concentrations of PCI or ProNtPCI were added and the measures were continued for 2 min. The slope of the first part of the assay corresponded to m o and the slope of the second part to m i . The residual enzymatic activity was calculated (m o to m i ) and plotted as function of the inhibitor concentration. Mass spectrometry Molecular mass was determined by MALDI-TOF mass spectrometry on a Bruker–Biflex spectrometer. Ionization wasaccomplishedwitha337-nmpulsednitrogenlaser and spectra were acquired in the linear positive ion mode, using a 19 kV acceleration voltage. Samples were pre- pared mixing equal volumes of the protein solution and a saturated solution of sinapinic acid, used as a matrix, in aqueous 30% acetonitrile with 0.1% trifluoroacetic acid (v/v). Circular dichroism spectroscopy CD spectra were collected on a Jasco-J715 spectropolari- meter at 25 °C, using a 2-mm path length cell, a band width of 2 nm, a step size of 0.5 nm and an averaging time of 1 s. Samples were analyzed in 0.1% trifluoroacetic acid (pH 2.0) or 50 m M Na 2 HPO 4 (pH 7.0), at 100 lgÆmL )1 final con- centration. Deuterium to proton (D/H) exchange Fifteen micrograms of lyophilized samples of PCI or ProNtPCI were resuspended in 5 lLofD 2 O and incubated for 3 h at 50 °C in order to exchange completely all labile protons and afterwards were maintained for 30 min at room temperature to refold properly. The native deuterated proteins were diluted with four volumes of 15 m M glycine pH 3.0 in H 2 O to start the hydrogen exchange and samples were taken in a time-course manner and analyzed by MALDI-TOF MS. Exoproteolysis Fifteen micrograms lyophilized aliquots of ProNtPCI were dissolved in 10 lL10m M Tris/HCl buffer (pH 8.5) con- taining 5 lg of leucine aminopeptidase (Sigma). Samples were collected in a time-course manner, diluted with water containing 0.1% trifluoroacetic acid (1 : 2) and the pro- teolyzed products present in the mixture were identified by MALDI-TOF mass spectrometry. Nuclear magnetic resonance NMR spectra were recorded on a Bruker AMX spectro- meter operating at 500 MHz. Two milligrams of PCI, 2 mg of the N-terminal pro-sequence peptide and 100 lgof ProNtPCI were resuspended in 500 lLofNaH 2 PO 4 pH 4.00 containing 10% D 2 O and the spectra were acquired at 35 °C. Results Expression in E. coli In order to study experimentally whether PCI pro-regions influence the folding of mature PCI, two precursor forms of PCI were obtained by recombinant expression. The first expression trials of ProNtPCI in E. coli MC1061 using a pINIIIOmpA3-derived secretion vector led to a low yield of purified protein due to the proteolysis of the pro-sequence during the protein expression period. The ProNtPCI protein wasdegradedtoPCIthataccumulatedintheculture medium. The final yield of intact ProNtPCI was very low (50 lgÆL )1 ) and it was used exclusively for the experiments requiring small amounts of protein. In another expression system ProNtPCI was cloned into pBAT4 vector with and without the signal peptide OmpA, to produce the protein either extracellularly or intracellularly in BL21 cells. The extracellular expression of the molecule resembled that of ProNtPCI in MC1061 cells. The intracellular expression led to the formation of inclusion bodies, probably due to the fact that PCI contains three disulfide bonds that can not be efficiently formed inside the reductive environment of the host cells, leading to the accumulation of protein aggregates. After purification and disaggregation of the inclusion bodies, the final yield of ProNtPCI produced with this system was 3.4 mgÆL )1 (see Experimental procedures). The same expression protocol was used for the intracellular expression of ProPCI, the other precursor form analyzed in the refolding studies, which gave a final yield of 3.0 mgÆL )1 of protein. Refolding in vitro Mature PCI refolding undergoes a two-stage process: a first stage of fast unspecific disulfide formation is followed by a second stage (rate limiting step) of disulfide reshuffling which leads to the native form [18]. Such behaviour was investigated for the different recombinant molecular forms of this study. The yield of native-like forms achieved after 7 h of refolding in absence of an external thiol was similar in all the molecules tested (<5%) (Fig. 1A, left). The RP-HPLC chromatogram profiles from the 7 h refolding mixture of Ó FEBS 2003 Folding of PCI precursor form (Eur. J. Biochem. 270) 3643 PCIandPCIplustheProNtintrans were indistinguishable. Thus, we can assume that the molar ratio among scrambled and native species is not affected by the addition of the ProNt segment in trans. The ratio between the native form and the ensemble of scrambled species remains constant for all tested forms. Therefore, in the absence of redox agents, neither the N-terminal nor the C-terminal PCI pro-sequences have an effect on the final yield of native PCI, indicating that the overall folding process is similar among all the molecules tested under these conditions. It is worth mentioning that the folding of PCI is accelerated by the presence of external thiols in the folding mixture [18]. While the addition of Cys accelerates the second stage of disulfide reshuffling of scrambled forms to native PCI, the addition of Cys-Cys enhances the first stage of disulfide formation, which leads to the formation of scrambled species. We analyzed the refolding process of all the above mentioned recombinant forms in the presence of 4 m M Cys and 2 m M Cys-Cys. The RP-HPLC profiles show that the folding kinetics and the fold- ing efficiency are higher under such conditions. The yield of native species achieved is superior to 70% after 1 h of refolding (Fig. 1B). The folding kinetics and efficiency of PCI and PCI plus ProNt peptide in trans are similar but, surprisingly, for ProNtPCI and ProPCI the folding kinetics are a little slower (Figs 1B and 2). Nevertheless, they can be considered to have a similar folding efficiency, as the final yields of native form at 24 h of refolding are nearly identical (Fig. 2). The flow of intermediate species containing one, two and three disulfides was followed by MALDI-TOF mass spectro- metry analyzing the iodoacetate-trapped folding inter- mediates in the four sets of tested molecules. The flow of refolding intermediates is characterized by a progression from the reduced state through the more thermodynam- ically stable 1-, 2- and 3-disulfide species. The rate of disulfide formation was similar in all the molecules tested under the same conditions. Influence of pro-sequences in vivo It has been reported that some mutations of mature PCI at the C-tail give rise to low expression yields or low folding efficiencies compared to wild-type mature PCI: D3, Y37G and G35P/P36G PCI [22]. To determine whether the N-terminal pro-region might improve their in vivo expres- sion or folding in E. coli, the expression of each PCI mutant protein and wild-type PCI was analyzed in parallel to the corresponding ProNtPCI mutant protein and wild-type ProNtPCI. Twenty-four hours after induction of protein expression the supernatant was collected, analyzed by RP-HPLC (Fig. 3) and the species were identified by Fig. 1. In vitro folding studies of PCI, PCI plus the ProNt in trans, ProNtPCI, and ProPCI in the presence of selected redox agents. Reduced and denatured proteins were refolded in the absence (no added thiols) (A), or presence (4 m M Cys/2 m M Cys-Cys) (B) of external thiols. Folding intermediates were acid-trapped and analyzed by RP-HPLC. Elution positions of native (N) and reduced (R) forms are indicated. Fig. 2. Refolding efficiencies of PCI, PCI plus the ProNt peptide in trans,ProNtPCIandProPCI.Reduced and denatured proteins were refolded in the presence of 4 m M Cys/2 m M Cys-Cys and acid-trapped folding intermediates were analyzed by RP-HPLC. The yield of native form was calculated in each time point from the peak areas in the corresponding RP-HPLC chromatograms. 3644 S. Bronsoms et al. (Eur. J. Biochem. 270) Ó FEBS 2003 MALDI-TOF mass spectrometry. As previously mentioned, theN-terminalpro-regionisdegradedintheE. coli extracellular media when secreted therefore the protein species found in the culture media were the mature PCI forms without the pro-region. The amount of each expressed protein was calculated by comparison of the corresponding RP-HPLC peak areas (data not shown). The final yield of each native-like ProNtPCI mutant, the ratio between the native form and the ensemble of scrambled species, and the ratio among the scrambled species present in the cell culture supernatants were compared with those of PCI mutants to evaluate any influence of the pro-region. Under the conditions of the experiment, the values obtained were similar for mature PCI and the ProNtPCI mutant proteins, indicating that the pro- region of PCI affects neither the expression levels nor the folding efficiencies in vivo in E. coli. Inhibitory activity To test whether ProNtPCI displays the same biological activity as mature PCI, inhibition studies of carboxypep- tidase A1 (CPA1) enzyme by ProNtPCI and PCI were performed. Both proteins show very similar affinities for CPA1 using the substrate furyl-acryloyl- L -phenylalanyl- L -phenylalanine and they have the same IC 50 value (100 n M ). According to these results, the mature PCI region within ProNtPCI should keep the same disulfide pairing and a similar three-dimensional structure as in isolated mature PCI, at least in the region which docks with the enzyme. Structural analyses Despite the small amounts of regular secondary structures of wild-type PCI native form [26], far-UV CD spectroscopy may be helpful to indicate its folding state, as it shows a characteristic positive ellipticity band at 228 nm when it is properly folded and possesses the wild-type Y37 residue [22]. Thus, this maximum band at 228 nm would also be expected for ProNtPCI. However, when the CD spectrum of ProNtPCI was recorded, such a spectral band was not observed at pHs of either 2.0 or 7.0 (Fig. 4). So, apparently, the environment of Y37 is affected in the pro form. Time-course deuteron to proton exchange monitored by MALDI-TOF MS [27] was also performed for both proteins. PCI contains 65 labile hydrogens and NMR has demonstrated that five of them form the slow exchange core [26]. The results indicate that the hydrogen exchange kinetics followed by both proteins are similar (Fig. 5). For each protein a major subpopulation of protons exchange rapidly (within 2 h) and the equilibrium is reached after 24 h. However, the number of slow exchanging deuterons is significantly different. While PCI retains five deuterons protected from exchange when equilibrium is reached, ProNtPCI retains nine under the same conditions. In addition, the number of deuterons retained before achieving Fig. 3. In vivo expression of recombinant forms of wild-type PCI, wild-type ProNtPCI and variants of them with mutations at the C-tail. (A) Schematic representation of the recombinant proteins produced for this study. Amino acids are in one-letter code. The N-terminal pro-sequence is indicated with a white box, the mature protein with a light shaded box and the mutated amino acids with a white box. (B) Recombinant proteins were produced in E. coli MC1061 cells in the expression vector pINIII-OmpA3. The supernatants, after 24 h induction, were analyzed by RP-HPLC. The quantity of each native and scrambled form was calculated from the peak area of its corresponding RP-HPLC chromatogram. The elution position of each native or native-like form is indicated (N). In case of G35P/P36G mutants the disulfide pairing is not the same as wild-type PCI [22]; in these cases, S stands for the most stable form. Ó FEBS 2003 Folding of PCI precursor form (Eur. J. Biochem. 270) 3645 the equilibrium is significantly higher in ProNtPCI than in mature PCI. These differences show either that ProNtPCI presents a different conformational state than PCI or that the N-terminal pro-sequence is structured and protects some protons from exchange. We have recently shown that exoproteolysis with leucine aminopeptidase followed by MALDI-TOF MS may pro- vide information about the occurrence of secondary struc- ture elements along proteins and about their stability [28]. Accumulation of certain stable protein fragments can be observed, which correspond to the beginning of the secondary structures present in the protein and thus, the identification of these fragments leads to a quick mapping of the regular secondary structures. When we applied this procedure to ProNtPCI two major stop points for proteo- lysis, which give rise to two accumulated protein fragments starting at positions 10a and 15a (in reference to the N-terminal propeptide) were identified along the N-terminal pro-sequence (Fig. 6). These two stop points are an indication of the presence of secondary structures in the N-terminal pro-region of ProNtPCI. Finally, NMR analyses of the N-terminal peptide, ProNtPCI and PCI were performed. The 1 H-NMR spectrum of the isolated (synthetic) N-terminal pro-peptide (Fig. 7A) does not show a large dispersion of resonances at the low or high fields. In the 0–1 p.p.m. region there are not a significant number of potential shifted methyl protons, and the dispersion of resonances is also small in the NH region (9–12 p.p.m.). The bidimensional TOCSY and NOESY proton NMR spectra of this molecule were also recorded (data not shown). Comparison of both spectra indicate the lack of non-sequential interactions and, hence, the lack of a compact and defined fold. The comparative analysis of 1 H-NMR spectra of mature PCI and ProNtPCI indicates that both molecules display a significant dispersion of resonances at both the low field (amide and aromatic region) and the high field (methyl region) (Fig. 7B,C), reflecting a well folded and tight globular structure, as recently we reported for mature PCI [26]. The ProNtPCI spectrum displays a noisier appearance than that of PCI due to the limited amount of protein available to perform the experiment. However, it can be observed that the pattern of resonances in both, amide and methyl regions, show important differences. Such differences could arise from changes in the structure of the PCI core and/or from the additional structure adopted by the pro-region and its interactions with the PCI core. Discussion Previous work from our laboratory has demonstrated that PCI can correctly refold in vitro with kinetics and efficiencies depending on the redox conditions used [18]. As its rate of refolding in vitro is extremely slow, other folding helpers, as molecular chaperones, isomerases or pro-regions are expec- ted to catalyze its folding in vivo. Both, its N- and C-terminal pro-regions are removed in vivo before the protein is secreted, so it is plausible that these extensions might be involved in the folding process. Such a role could be assigned to the Fig. 5. Kinetic plots of the D/H exchange monitored by MALDI-TOF MS. The decrease in deuteration levels of PCI (n)andProNtPCI(e) were measured after dilution (1/5) of the deuterated samples in a proton buffer (15 m M glycine, pH 3.0). Fig. 4. Circular dichroism studies. CD analyses of native PCI and ProNtPCI were carried out in 20 m M phosphate buffer (pH 7.0) and 0.1% (v/v) trifluoroacetic acid (pH 2.0); 100 lg of protein were used in each spectrum. 3646 S. Bronsoms et al. (Eur. J. Biochem. 270) Ó FEBS 2003 N-terminal pro-sequence, in a first instance, due to its very long size (two thirds of the mature protein) and also due to proof that the C-terminal pro-sequence probably is involved in sorting to vacuoles [17]. In this work, the in vitro folding of mature PCI has been compared with that of ProNtPCI, ProPCI and PCI with the ProNt in trans. In the tested conditions, the kinetic rates for PCIplustheProNtintrans are similar to those of PCI, but the kinetic rates for ProNtPCI and ProPCI are slightly slower. It is surprising that the presence of the pro-sequence extensions in cis causes a slight decrease on the overall folding kinetics. Nevertheless, neither the progression of disulfide intermediates nor the final percentage of native form achieved (folding efficiency) is altered. Therefore, in contrast to our initial expectations, we can conclude that neither the N-terminal nor the C-terminal pro-sequence appears to have a positive effect on the efficiency and the mechanism of PCI folding. Similar results have been reported in the folding studies of another small disulfide- rich protein, the x-conotoxin [13,14], where the N-terminal pro-sequence has no effect on the mature protein folding. In contrast, for this protein, it has been found that the presence of an additional glycine residue at the C terminus (equi- valent to the C-terminal pro-sequence) enhances the yield of properly folded x-conotoxin MVIIA. It is noteworthy that a somewhat similar glycine residue, placed after the last cysteine of the T-knot core, is conserved among all the known members of the squash inhibitor family and in PCI as well, where it plays an important role on its folding [22]. Unlike the case of conotoxins, in the latter molecules this glycine cannot be ascribed to the C-terminal pro-sequence, as it is kept in the protein after maturation. The in vivo studies presented here show that for PCI and for the mutant proteins tested the N-terminal pro-sequence has no effect on the expression level or the final yield of native form produced. In addition, the ratio between the scrambled species and the native form that appears in the cell culture supernatants were similar. Thus, the N-terminal pro-region does not affect the in vivo folding of the mature PCI in E. coli either. However, it could be argued that in its biological environment, in potato, the pro-region of PCI could play a role in the in vivo folding, interacting with other cellular factors. A slightly different behaviour has been described for another small disulfide-rich protein, BPTI, in which the N-terminal pro-region has not been found to play a substantial role in its disulfide bond formation or rearrangement within microsomes [11], even though it seems to have a slight positive effect on its folding rate in vitro [10]. We have investigated the presence of ordered structural elements in the N-terminal pro-region of PCI by several approaches. In the CD experiments it was observed that the characteristic positive ellipticity band of PCI at 228 nm disappears in ProNtPCI, despite it displays CPA inhibitory activity and contains the Y37 residue, which seems to contribute to such a band [22]. The presence of new structural elements in the N-terminal pro-region could modify the signal in this region and mask the characteristic maximum at 228 nm of wild-type PCI, giving rise to the spectrum observed for ProNtPCI. It has been proposed that the last few amide hydrogens to exchange in a protein constitute the slow exchange core, that is formed by the secondary structure elements that are more tightly packed in a protein [29,30]. Given Fig. 6. Exoproteolysis of ProNtPCI with leucine aminopeptidase. (A) Lyophilized protein (15 lg) was proteolyzed with Leu aminopeptidase and samples collected in a time-course manner were analyzed by MALDI-TOF mass spectrometry [29]. Left, 1 h exoproteolysis sample; right, 3.5 h exoproteolysis sample; y axis, spectral intensity arbitrary units; x axis, mass/charge ratio. Inner box, correspondence between molecular mass of the visualized fragments and predicted stop points of proteolysis along the sequence. (B) Schematic representation in one-letter amino acid code of ProNtPCI, with shaded arrows indicating accumulated peptides from the Leu aminopeptidase proteolysis reaction. Ó FEBS 2003 Folding of PCI precursor form (Eur. J. Biochem. 270) 3647 that the number of slow exchanging protons of ProNtPCI was significantly higher than that of PCI, we can assume that this molecule displays additional structural elements in comparison to PCI. The leucine aminopeptidase exoproteolysis experiments followed by MS also bore evidences that the N-terminal pro-region contains secondary structure. Interestingly, the presence of two major stop points of exoproteolysis in the Fig. 7. NMR spectra of the isolated N-terminal pro-segment (A), mature PCI (B) and the ProNtPCI form (C). Two milligrams of PCI and of isolated N-terminal pro-sequence and 100 lg of ProNtPCI were dissolved in 500 lLof20m M NaH 2 PO 4 at pH 4.00, containing 10% D 2 O and the spectra were recorded at 35 °C in a 500-MHz spectrometer. Insets show expanded high and low field areas of the spectra of the proteins. The strong resonances at around 1.9, 2.1 and 8.6 p.p.m., visualized in the spectrum C, are attributed to organic molecule contaminants. 3648 S. Bronsoms et al. (Eur. J. Biochem. 270) Ó FEBS 2003 center of ProNtPCI pro-region gives us information about the possible boundaries of secondary structure elements. As we have shown recently [28], such a behaviour is found to be generated by the initial residues of stable regular secondary structure elements in globular proteins, when these ones are trimmed by exoproteases. In depth NMR analysis (i.e. n-dimensional) of ProNtPCI has not been performed due to the small amount of material available. However, the comparison of monodimensional 1 H-NMR spectra indicates that ProNtPCI has a well- defined three-dimensional globular structure and that displays some extra interactions in addition to those belonging to the mature native PCI. At this respect, are noteworthy the changes observed at very low field (amide region) between the spectra of both proteins, and parti- cularly the resonance visualized at about 11.8 p.p.m. for ProNtPCI, not present for wild-type PCI. Given that the N- and C-terminal pro-regions do not appear to play a substantial role in PCI folding, either in vitro or in vivo in E. coli, which role could be proposed for them? The pro-region of acetylcholine esterase in Pichia pas- toris modulates the protein secretion [31] and the pro-region of caspase-8 interacts with the tumor necrosis factor receptor [32]. Similarly, PCI pro-regions could be involved in targeting the molecule within the cell or in modulating the interactions with other proteins or biomolecules. Acknowledgments This work was supported by grant BIO2001-2046 from MCYT (Ministerio de Ciencia y Tecnologı ´ a, Spain) and by the Centre de Refere ` ncia en Biotecnologia de la Generalitat de Catalunya. S. 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