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

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

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
native-like 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
intra-molecular chaperones, 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 the rate of the folding process in 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,

08193Bellaterra (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 carbcarb-oxypeptidase

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)

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 XbaI and EcoRI and ligated into

pIN-III-OmpA3vector 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 pINIIIOmpA3vector, E coli

MC1061 cells were grown at 37C and expression was

induced at 0.1 attenuation unit at 550 nm by the addition of

1 mM isopropyl thio-b-D-galactoside, and they were

har-vested by centrifugation (13000 g at 4C for 45 min) 20 h

after induction of protein expression ProNtPCI was

purified from the culture medium by a Sep-Pak C18

(Waters) cartridge and eluted with 70% isopropanol

containing 0.1% trifluoroacetic acid The protein was

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 mM

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 mM Tris/HCl, 0.5 mM 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 mMTris/HCl, 0.5 mMEDTA and 2% Triton X-100 (pH 8.5) and centrifuged at 22 000 g for

25 min The pellet was resuspended in 10 mL 6M guani-dinium chloride and 30 mMdithiothreitol (pH 8.5) After

6 h the sample was centrifuged at 3000 g for 10 min and the supernatant was dialyzed against 0.1M Tris/HCl (pH 8.5) for 12 h and then renaturation was performed

by dialysis in the presence of a redox system containing

4 mMCys and 2 mMCys-Cys (cystine) at pH 8.5 for 48 h

at 4C 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 (0.5M, pH 8.5) containing 5Mguanidinium chloride and

3 0 mM dithiothreitol, to a final protein concentration of 46.5 lM After 2 h at 25C, the reduced and denatured proteins were passed through a PD-10 (Pharmacia) column equilibrated with 0.1M 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 lM, with the 0.1M Tris/HCl buffer (pH 8.5), the same buffer containing 1 mMCys and the same buffer containing 4 mM

Cys and 2 mM 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 Samples

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) 0.1Miodoacetic acid

in Tris/HCl buffer (0.5M, pH 6.5) containing 40% (by

volume) of dimethylformamide (irreversible trapping) [25]

Carboxymethylation was allowed to proceed for 30 min at

25C

Inhibitory activity

The substrate used to perform the carboxypeptidase

activity was 0.2 mMfuryl-acryloyl-L-phenylalanyl-L

-phenyl-alanine and the buffer was 50 mM Tris/HCl, 0.5MNaCl,

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 lL of increasing

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 mo and the slope

of the second part to mi The residual enzymatic activity

was calculated (mo to mi) 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

was accomplished with a 3 3 7-nm pulsed nitrogen laser

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 25C, 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 mM Na2HPO4 (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 lL of D2O and incubated

for 3h at 50C 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 mMglycine

pH 3.0 in H2O 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 lL 10 mM 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 lg of ProNtPCI were resuspended in 500 lL of NaH2PO4

pH 4.00 containing 10% D2O and the spectra were acquired at 35C

Results Expression inE 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 was degraded to PCI that accumulated in the culture 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

Refoldingin 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

PCI and PCI plus the ProNt in trans 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 mMCys and 2 mMCys-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-sequencesin 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 expresexpres-sion 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, ProNtPCI and ProPCI 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.

MALDI-TOF mass spectrometry As previously mentioned,

the N-terminal pro-region is degraded in the E 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 IC50value (100 nM)

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.

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 1H-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 of1H-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) and ProNtPCI (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 dichroismstudies 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.

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

PCI plus the ProNt in trans 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.

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 lL of 20 m 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.

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

1H-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 vitroor in vivo in E coli, which role could be proposed for

them? The pro-region of acetylcholine esterase in Pichia

pas-torismodulates 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 Bronsoms is a predoctoral fellowship recipient from the Generalitat

de Catalunya.

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