unfolding transitions of bacillus anthracis protective antigen

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Unfolding transitions of Bacillus anthracis protective antigen David A. Chalton a , Ian F. Kelly a , Alistair McGregor a , Helen Ridley a , Allan Watkinson b , Julie Miller c , Jeremy H. Lakey a, * a Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle NE2 4HH, UK b Avecia (Biotechnology) P.O. Box 2, Belasis Avenue, Billingham, Cleveland TS23 1YN, UK c Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK Received 13 March 2007, and in revised form 24 April 2007 Available online 11 May 2007 Abstract Protective antigen (PA) is an 83 kDa protein which, although essential for toxicity of Bacillus anthracis, is harmless and an effective vaccine component. In vivo it undergoes receptor binding, proteolysis, heptamerisation and membrane insertion. Here we probe the response of PA to denaturants, temperature and pH. We present analyses (including barycentric mean) of the unfolding and refolding behavior of PA and reveal the origin of two critical steps in the denaturant unfolding pathway in which the first step is a calcium and pH dependent rearrangement of domain 1. Thermal unfolding fits a single transition near 50 °C. We show for the first time circular dichro- ism (CD) spectra of the heptameric, furin-cleaved PA63 and the low-pH forms of both PA83 and PA63. Although only PA63 should reach the acidic endosome, both PA83 and PA63 undergo similar acidic transitions and an unusual change from a b II to a b I CD spectrum. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Protein refolding; Bacillus anthracis; Vaccine; Protein structure; Fluorescence; Circular dichroism; Differential scanning calorimetry; Baryc- entric mean The 83 kDa anthrax protective antigen (PA83) 1 is non- toxic but essential for the toxic activity of Bacillus anthracis the causative agent of anthrax. It binds to surface receptors [1,2] on mammalian cells before undergoing furin depen- dent cleavage which creates PA20 and PA63 fragments. PA20 (amino acids 30–196 of the immature PA) forms part of the N-terminal domain-1 (amino acids 30–287 of the immature PA) of this four-domain protein. PA63 remains bound to the receptor, heptamerises and combines with a mixture of up to three lethal factor (LF) or edema factor (EF) proteins [3–5] to create a number of different but func- tionally similar complex toxins [6,7]. After receptor-medi- ated endocytosis, the role of PA is to translocate these two actively toxic proteins into the cytoplasm of target cells. This function is facilitated by a PA63 membrane insertion step which occurs in the acid environment of the late endosome [8,9]. As its name suggests PA has been used to raise protective antibodies against anthrax infec- tion and this is possible because it is non-toxic and thought to be essential for infection [10]. Recently, however some toxin deficient strains have been shown to cause lethal pul- monary anthrax infection similar to the parental strains although with different histopathology [11]. Like many bacterial toxins PA is secreted across the membrane of the B. anthracis producing-cell as a water soluble monomer [7]. Thus, during its active life, the PA molecule undergoes (i) a critical post-secretion folding step within the B. anthra- cis cell wall, (ii) a post-cleavage oligomerisation and refold- ing at the target cell surface and (iii) a pH induced conformational change in the endosome [12]. This pH 0003-9861/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.04.030 * Corresponding author. Fax: +44 191 222 7424. E-mail address: [email protected] (J.H. Lakey). 1 Abbreviations used: PA, B. anthracis protective antigen; PA83, recombinant PA; PA63, recombinant furin-cleaved PA83 large fragment; PA20, recombinant furin-cleaved PA83 small fragment; Apo-PA, PA83 lacking calcium ions; ICP-AA, inductively coupled plasma atomic absorption spectroscopy. www.elsevier.com/locate/yabbi ABB Archives of Biochemistry and Biophysics 465 (2007) 1–10

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www.elsevier.com/locate/yabbi

ABBArchives of Biochemistry and Biophysics 465 (2007) 1–10

Unfolding transitions of Bacillus anthracis protective antigen

David A. Chalton a, Ian F. Kelly a, Alistair McGregor a, Helen Ridley a,Allan Watkinson b, Julie Miller c, Jeremy H. Lakey a,*

a Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle NE2 4HH, UKb Avecia (Biotechnology) P.O. Box 2, Belasis Avenue, Billingham, Cleveland TS23 1YN, UK

c Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK

Received 13 March 2007, and in revised form 24 April 2007Available online 11 May 2007

Abstract

Protective antigen (PA) is an 83 kDa protein which, although essential for toxicity of Bacillus anthracis, is harmless and an effectivevaccine component. In vivo it undergoes receptor binding, proteolysis, heptamerisation and membrane insertion. Here we probe theresponse of PA to denaturants, temperature and pH. We present analyses (including barycentric mean) of the unfolding and refoldingbehavior of PA and reveal the origin of two critical steps in the denaturant unfolding pathway in which the first step is a calcium and pHdependent rearrangement of domain 1. Thermal unfolding fits a single transition near 50 �C. We show for the first time circular dichro-ism (CD) spectra of the heptameric, furin-cleaved PA63 and the low-pH forms of both PA83 and PA63. Although only PA63 shouldreach the acidic endosome, both PA83 and PA63 undergo similar acidic transitions and an unusual change from a b II to a b I CDspectrum.� 2007 Elsevier Inc. All rights reserved.

Keywords: Protein refolding; Bacillus anthracis; Vaccine; Protein structure; Fluorescence; Circular dichroism; Differential scanning calorimetry; Baryc-entric mean

The 83 kDa anthrax protective antigen (PA83)1 is non-toxic but essential for the toxic activity of Bacillus anthracis

the causative agent of anthrax. It binds to surface receptors[1,2] on mammalian cells before undergoing furin depen-dent cleavage which creates PA20 and PA63 fragments.PA20 (amino acids 30–196 of the immature PA) forms partof the N-terminal domain-1 (amino acids 30–287 of theimmature PA) of this four-domain protein. PA63 remainsbound to the receptor, heptamerises and combines with amixture of up to three lethal factor (LF) or edema factor(EF) proteins [3–5] to create a number of different but func-

0003-9861/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.abb.2007.04.030

* Corresponding author. Fax: +44 191 222 7424.E-mail address: [email protected] (J.H. Lakey).

1 Abbreviations used: PA, B. anthracis protective antigen; PA83,recombinant PA; PA63, recombinant furin-cleaved PA83 large fragment;PA20, recombinant furin-cleaved PA83 small fragment; Apo-PA, PA83lacking calcium ions; ICP-AA, inductively coupled plasma atomicabsorption spectroscopy.

tionally similar complex toxins [6,7]. After receptor-medi-ated endocytosis, the role of PA is to translocate thesetwo actively toxic proteins into the cytoplasm of targetcells. This function is facilitated by a PA63 membraneinsertion step which occurs in the acid environment ofthe late endosome [8,9]. As its name suggests PA has beenused to raise protective antibodies against anthrax infec-tion and this is possible because it is non-toxic and thoughtto be essential for infection [10]. Recently, however sometoxin deficient strains have been shown to cause lethal pul-monary anthrax infection similar to the parental strainsalthough with different histopathology [11]. Like manybacterial toxins PA is secreted across the membrane ofthe B. anthracis producing-cell as a water soluble monomer[7]. Thus, during its active life, the PA molecule undergoes(i) a critical post-secretion folding step within the B. anthra-

cis cell wall, (ii) a post-cleavage oligomerisation and refold-ing at the target cell surface and (iii) a pH inducedconformational change in the endosome [12]. This pH

2 D.A. Chalton et al. / Archives of Biochemistry and Biophysics 465 (2007) 1–10

dependent structural change produces a 14-stranded trans-membrane b-barrel in which a ring of phenylalanines act asa translocation gate for the LF and EF toxins [4]. Further-more in relation to its role as an antigen, the intracellulardegradative pathway of PA is fundamental to vaccine effi-cacy (see below) [13] where it shows complex behavior [14].

The crystal structure of PA has been solved [15] andrevealed a complex four-domain protein analogous toother oligomerising toxins such as a hemolysin, leukocidinsand aerolysin [7]. Each of the steps in the action of PA(receptor binding, proteolytic activation, oligomerisationand endosome acidification) involves a subset of thedomains. Thus domain 4 is responsible for receptor bind-ing [16], domain 1 furin-cleavage and calcium binding[17], and domains 2 and 3 membrane insertion and oligo-merisation [18]. The final heptameric protein translocatingpore is made up of seven b-hairpins, one donated fromeach monomer.

The manufacture of recombinant PA (PA83) in Esche-

richia coli, by heterologous expression from a gene withaltered codons, avoids using the pathogen B. anthracis,and has proven to be very effective for vaccine use [10].This expression system produces recombinant PA83 asinclusion bodies which are then refolded from a urea dena-tured state. Thus the post-secretion, chaperone assistedfolding of PA83 in the B. anthracis cell wall [19,20] isreplaced by an in vitro urea dilution step. The structureof PA has been shown to strongly influence the mode ofantigen presentation to T-cells. Some surface exposedCD4 T-cell epitopes are effectively processed at the cellmembrane or in early endosomes whilst the majority,which are in folded domains, require the complete denatur-ation of the protein and processing in lysosomes [13].Mouse monoclonal antibodies, capable of neutralizingPA83 in vitro, target an exposed surface epitope [21,22].Hence, the folding and unfolding of PA83 is currently ofgreat interest since production of a stable, effective and safevaccine against B. anthracis infection is of increased impor-tance [23]. Two recent papers have studied the unfolding ofwild type (WT) PA and the role of calcium in the proteinstabilization [24,25] and this paper builds on their work.

Here we present an extensive analysis of the unfolding andrefolding behavior of PA and reveal the origin of two criticalsteps in the denaturant unfolding pathway. Furthermore, bycomparing PA83 with PA63 over a range of pH we show thatthe presence of domain 1 has little effect on the structuraltransition in the acidic endosome. This indicates that mixedPA63 and PA83 heptamers suggested recently may be able toundergo the correct pH transition [26].

Experimental procedures

All chemicals were bought from Sigma (Poole, UK) unless otherwisestated. Purified PA83 was a kind gift from Dstl, (UK) and Avecia, (UK),and was expressed in and purified from E. coli as previously described [10].PA63 was prepared from PA83 as described by [27] except that a QSepharose HR column replaced the Q Sepharose column and size exclu-sion chromatography steps. Analytical size exclusion chromatography was

performed using a Sephacryl S100 column, in 200 mM phosphate bufferpH 7.4.

Production of Apo PA83

PA83 (2.0 mg/ml) was prepared in 8 M urea, 150 mM NaCl, 10 mMMOPS (pH 7.4). This was dialysed using a 3500 MWCO membrane(SpectraPor, USA) against the same buffered urea solution supplementedwith 2 g/l Chelex-100 resin (Bio-Rad, USA) at room temperature for>24 h. The unfolded PA was then dialysed extensively against 150 mMNaCl, 10 mM MOPS (pH 7.4), 2 g/l Chelex-100, at 4 �C. The urea-freedialysis step refolded the protein with no visible aggregates. The calciumcontent of the refolded Apo-PA83 was determined by ICP-AAS to be lessthan one calcium ion per 10 monomers in a solution of 50 lM Apo-PA83.Protein concentrations were determined from UV absorption at 280 nmwith calculated extinction coefficients.

Unfolding–refolding PA83 with urea

Circular dichroismFar-UV CD spectroscopy (250–190 nm) was carried out on a Jasco J-

810 spectropolarimeter with bandwidth set to 2 nm and scanning speed of50 nm/min, using 0.2-mm-path length demountable cuvettes. Sampleswere examined at 25 �C in 20 mM sodium phosphate (BDH, UK) buffer,unless otherwise stated. For urea unfolding experiments PA83 was incu-bated at room temperature for more than 16 h at each urea concentrationwith 30 mM MOPS pH 8.0, and the CD measured at 220 nm. The fractionof PA83 unfolded (fu) at x M urea was calculated with the followingequation:

fu ¼V x � V f

V u � V f

ð1Þ

where Vu represents the far-UV CD value at 220 nm for unfolded PA83 in8 M urea, Vf for folded PA83 in 0 M urea, and Vx for PA83 in x M urea.Near-UV CD spectra (320–250 nm) were measured over a 1 cm pathlength with bandwidth set to 1 nm.

Intrinsic fluorescenceFluorescence measurements were made using a Varian Eclipse spec-

trofluorometer at 20 �C, using a bandwidth of 5 nm, a 2 nm/s scan rateand emission photomultiplier voltage of 600 V using a 5 mm-pathlengthcuvette (Hellma 101.057).

To measure unfolding, samples of PA83 were equilibrated in 30 mMMOPS with variable urea concentrations (ACS reagent) for more than12 h at room temperature. Experiments were performed in duplicate andthe mean value shown. For each sample, 25 lg (0.3 nmol) of denaturedprotein was diluted into 500 ll of an appropriate 30 mM MOPS/ureabuffer, and emission measured between 295 and 450 nm following exci-tation at 280 nm. A suitable blank was subtracted and the barycentricmean (mean of the integral between 295 and 450 nm) was calculated as

km ¼P

F ðkÞ � ðkÞP

F ðkÞ ð2Þ

where F(k) is the point fluorescence at wavelength k [28].Sigmoidal curves were fitted to the urea unfolding and folding data

using Origin 7.0 (OriginLab Corp, MA) and this was used to define themid-point of each unfolding step. Free energy of unfolding in the absenceof denaturant was estimated by extrapolation using the linear least squaresmethod described [29]. To measure refolding, PA83 was incubated in30 mM MOPS pH 7.0 � 8 M urea (ACS quality) for >6 h. Samples ofPA83 (25 lg; 0.3 nmol) were then diluted into 500 ll, 30 mM MOPS/ureabuffer for >12 h and the intrinsic fluorescence barycentric mean measuredas above.

Differential scanning calorimetryDifferential Scanning Calorimetry (DSC) was performed using a

Micro-Cal VP-DSC (MicroCal Europe, Milton Keynes, UK) (cell volume

D.A. Chalton et al. / Archives of Biochemistry and Biophysics 465 (2007) 1–10 3

0.52 ml). Samples were scanned with a scan rate of 1 �C/min, and a fil-tering period of 16 s with protein at 0.75 mg/ml and an appropriate bufferin the reference cell. All solutions were gently degassed prior to use. Datawere corrected for the buffer baseline (buffer scans performed underidentical conditions), and analyzed using standard MicroCal ORIGIN V.7software.

Effect of pH on the structure of PA83 and PA63

Circular dichroismFor variable pH experiments PA was added from a stock of 22.9 lM to

a series of buffers over a range of pH, to a final concentration of 6.0 lM.The following buffers were used at a final concentration of 50 mM, with150 mM NaCl; pH 2 to 3.5 glycine–HCl; pH 4 to pH 5.5, citric acid–tri-sodium citrate; pH 6 to pH 8, sodium phosphate; pH8.5 to pH 10 Tris–HCl. The buffers were pre-equilibrated to 20 �C, the temperature at whichthe CD measurements were taken.

FluorescencePA83 (0.61 lM) was incubated at the desired pH for 1 h, after which

time an intrinsic fluorescence measurement was made. 1-anilino-8-nap-thalene sulfonate (ANS) was added to an identical PA sample (5 lM) to afinal concentration of 60 lM, and ANS fluorescence was measured after1 h. The excitation wavelength used for intrinsic fluorescence was 280 nm,and 370 nm for ANS fluorescence, with excitation and emission band-widths of 5 nm. Samples were prepared and analyzed in triplicate at 20 �C.

To measure urea unfolding at low pH, fluorescence measurements weremeasured as before but wavelength emission maxima kmax were plottedwith normalized fluorescence emission intensity. The pH 7.0 and pH 4.0urea denaturations were carried out in 20 mM sodium phosphate, pH 7.0,or in 20 mM sodium acetate pH 4.0 with urea to the stated concentration.Initial PA83 concentration was 1.5 mg/ml, and 25 lg of PA83 was dilutedinto 500 ll of buffer.

To measure the effect of calcium upon the final stage of unfolding orrefolding, samples of PA83 were unfolded in �8 M urea for >6 h, aliquotswere taken and diluted into 30 mM MOPS pH 7.0 with or without 5 mMCaCl2 for >12 h, intrinsic fluorescence was measured as before.

Results

Unfolding and refolding of PA83 in urea

Mature PA83 and PA63 contain 7/28 and 4/21 trypto-phan/tyrosine residues respectively but as expected, intra-molecular energy transfer causes the fluorescence emissionto be dominated by tryptophan. The calcium binding site iscontained within the region aa 165–258, and therefore pres-ent and functional in PA63 [15]. The intrinsic fluorescence ofPA83 at pH 7.0 shows a biphasic intensity and wavelengthresponse to increasing concentrations of urea (Fig. 1a). Itis thus possible to follow unfolding by both intensity andwavelength shifts. The large wavelength changes promptedus to use barycentric mean wavelength (km) as an accuratemeasure of unfolding since, unlike intensity, it is unaffectedby experimental effects such as noise in the emission peaks(kmax) and inaccuracies in protein concentration (intensity)(Fig. 1b). With calcium-free (Apo-) PA83 the initial unfold-ing transition is lost but an identical transition is seenbetween 3 and 5 M urea (Fig. 1c) [24]. The effect of increasingurea concentration was also followed by circular dichroism(Fig. 2). Since the transitions appear to be due to separateevents and are reversible we have analyzed each using a

two state model. This is probably more true of the sharp fluo-rescence transitions which have their origins limited to tryp-tophan rich areas compared to the CD which reports on allfour domains.

The first structural transition occurs at 0.81 M urea byfluorescence (DGfold = 3.05 ± 0.15 kcal mol�1; m = 3.6kcal mol�1 M�1) and 0.64 M urea by far UV-CD(DGfold = 1.45 ± 0.15 kcal mol�1; m = 1.74 kcal mol�1

M�1). This can clearly be seen in Figs. 1b and 2 where thereis a red shift in the km, of approximately 10 nm and achange in far-UV CD indicative of the destabilization ofthe folded state corresponding to �40% of the total.Although unfolding of PA83 has been measured previouslyby CD and fluorescence, the current data is the first toclearly show both phases clearly defined by the two tech-niques [25]. Similar transitions were obtained when usingguanidine hydrochloride as a denaturant (data not shown).This indicates that the observed unfolding of the proteinwas a result of the denaturant not an artefact of urea-induced carbamylation of the protein leading to a changein secondary and tertiary structure [30].

The PA83 fluorescence is relatively insensitive to furtherincreases in denaturant concentration until a value of3.5 M urea is reached (Fig. 1b), when a further transitionoccurs with a mid-point at 4.1 M [urea] (DGfold = 4.93 ±0.24 kcal mol�1; m = 1.28 kcal mol�1 M�1). Here the km

red shifts by approximately 5 nm. Above 5 M urea, PA83fluorescence shows little further change. The second transi-tion is also observed by far-UV CD at 220 nm [24] (Fig. 2),however, compared to that observed by fluorescence, thisbegins at a lower concentration with a mid-point at2.8 M [urea] (DGfold = 3.6 ± 0.23 kcal mol�1; m = 1.42mol�1 M�1).

The sharper transition determined by intrinsic fluores-cence probably results from its origin in one or more coop-eratively folded regions with high tryptophan content,whereas far-UV CD reports on every peptide bond in thismulti-domain protein.

The refolding of PA83 (Fig. 1b) exactly follows theunfolding pathway, confirming that the unfolding andrefolding data were collected at equilibrium. As observedin the unfolding experiments, a structural change occurswhen refolding at concentrations between 5 and 3 M ureawith a mid-point of 4.05 M (DGfold = 4.8 ± 0.23 kcalmol�1). Similarly to observations in the unfolding experi-ments, between 3 and 2 M urea the fluorescence is againinsensitive to the concentration of urea.

DG experiments usually have a range of 5–15 kcal mol�1

so the second transition is at the lower end of these valueswhilst the Ca2+-dependent transition is much smaller andthus quite unlike a ‘‘normal’’ protein folding transition.It is thus likely to be the disruption of the domain 1 inter-face rather than a full unfolding. The low value for the 2ndtransition probably reflects the multi-domain poorly coop-erative transition.

A second transition occurs when the urea concentrationis reduced to less than 1 M, and there is a shift of the km of

Fig. 1. (a) Intrinsic fluorescence of PA83 at a range of urea concentrations. Urea denaturation was performed in 20 mM sodium phosphate, pH 7.0, withurea at the stated concentration for 24 h. Initial PA83 concentration was 1.5 mg/ml, and a final concentration of 50 lg/ml, was used. Fluorescencemeasurements were at 20 �C, using a bandwidth of 5 nm, a 2 nm/s scan rate and a 5 mm-pathlength cuvette (Hellma) following excitation at 280 nm. (b)Unfolding and refolding of PA83 in the presence of various concentrations of urea. Fluorescent measurements were made using a Varian EclipseSpectrofluorometer at 20 �C. Unfolding: using 25 lg of PA83 in 30 mM MOPS/urea. Refolding: 25 lg of denatured PA83 (�8 M urea >6 h) was dilutedinto an appropriate 30 mM MOPS/urea buffer for >12 h. Supplementing denatured PA83 in 30 mM MOPS pH 7.0 with 5 mM CaCl2 for >24 h, leads to ashift in the Barycentric mean which is much closer (within 0.3 nm) to that of non-denatured PA83 (delimited by arrows and hatched area). (c) Properties ofthe intrinsic fluorescence of PA and ApoPA upon unfolding with urea. PA83 (50 lg/ml) was incubated for 24 h in 20 mM sodium phosphate, pH 7.0, withan appropriate concentration of urea. Fluorescence measurements were made as per (a). Apo PA83 (50 lg/ml) was incubated >12 h at room temperaturein 30 mM MOPS pH 7.0, 150 mM NaCl, 5 mM EDTA at a range of urea concentrations. The barycentric mean emission wavelength (calculated between295 and 450 nm) was used as it was more informative than normalized fluorescence intensity (normalized at 0 M urea).

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approximately 5 nm. This shift however does not return thekm to the original value (343 nm), despite only 13 mM ureapresent (due to limits of dilution). As can be seen from theunfolding experiments this wavelength value (347 nm)

occurred at approximately 1 M urea in the unfoldingexperiments (Fig. 1b). Apo-PA83 in which calcium has beencompletely removed displays a value of 352 nm at zero urea(Fig. 1c). Addition of 5 mM CaCl2 to the refolding

Fig. 2. Unfolding of PA83 in the presence of increasing concentrations ofurea. PA83 was incubated at room temperature >16 h in variousconcentrations of 30 mM MOPS supplemented with urea to specifiedconcentration, and the Far-UV CD220 at 20 �C was followed. The CD220

value for 8 M urea was assumed to represent fully unfolded PA83, and theCD220 value for 0 M urea was assumed to represent fully folded PA83.

D.A. Chalton et al. / Archives of Biochemistry and Biophysics 465 (2007) 1–10 5

buffer allowed PA83 to refold to a native-like state (asrevealed by intrinsic fluorescence) with a barycentric meanwithin 0.3 nm of the native PA83 region (Fig. 1b indicatedby hatched area and delimited by arrows). Thus in the pres-ence of calcium the folding is entirely reversible, this occur-rence is simplified by the absence of cysteine residues inPA83, removing the complication of mixed disulphide bondformation.

Unfolding of PA83 and PA63 at acidic pH

We incubated PA83 at various pH values for >12 hprior to recording the effect on structure by far-UV circulardichroism (Fig. 3b) and near-UV circular dichroism(Fig. 3b inset).

Buffer constituents were appropriate for the pH rangebeing investigated and they possessed buffering capacity,despite differing constituents their concentrations and thetypes of buffer used are unlikely to have other effects suchas ionic strength influences.

PA83 was only incubated for 1 h at pH 4.0 due to theaggregation if incubated for a longer time. We observed lit-tle change in structure between pH 6 and pH 10 by eithertechnique (data not shown), however there were largechanges in both intrinsic and ANS fluorescence (Fig. 3a)and circular dichroism (Fig. 3b and c) below pH 6. Theprotein aggregates between pH 5.0 and pH 4.0 whichapproximates to the values obtained in the endosome.PA83 displays a non-classical bII spectrum with a minimumat 208 nm and a zero value at 194 nm (Fig. 3b). This is rem-iniscent of porcine elastase (PDB code 1QNJ [31]) a mem-ber of the bII classification of b sheet rich proteins whichhave sufficient amounts of polyproline II (PPII) structureto obscure the weaker b CD signal [32]. Interestingly bothporcine elastase and PA contain large amounts of non-sec-

ondary (loop) structure. However, below pH 4.0 the pro-tein adopts a stronger more predominantly b-strand (bI)spectrum suggesting a possible unstructured or PPII to b-transition (Fig. 3b). The far-UV CD spectrum of PA83 isalso shown in phosphate buffer and a short pathlength cuv-ette to obtain shorter wavelength data in Fig. 4a.

The increase in ANS binding at low pH is indicative ofexposed hydrophobic patches and a molten globule state(supported by the reduction of near UV-CD signal(Fig. 3b inset)) both of which are associated with mem-brane insertion competent forms [33].

The PA63, formed by treatment with furin was shown toform the heptameric oligomer form by size exclusion chro-matography (VE of PA83 = 42.7 ml, whereas heptamericPA63 eluted at 35.3 ml. BSA (66 kDa) and Beta-amylase(200 kDa) standards eluted at 43.9 and 37.3 ml, respec-tively). Ion-exchange purified PA63 penetrated a 12%native gel but failed to migrate within it. Additionally, a12% SDS–PAGE analysis of glutaraldehyde-crosslinkedPA63 oligomer formed a >250 kDa tightly resolved band,whilst a non-crosslinked sample resolved at the expected63 kDa (data not shown).

This oligomer was analyzed by far-UV CD spectroscopy(Fig. 3c) which revealed a spectrum similar to that of PA83,in agreement with the published X-ray structure [5]. ThepH transition as measured by CD is surprisingly similarto that observed with monomeric PA83 suggesting thatthe pH induced structural changes in the monomer andheptamer are similar.

In an attempt to quantify the secondary structuralchanges due to pH we first compared the secondary structureof the X-ray derived models with the results from a webbased analysis of the far UV-CD data. The known secondarystructure content calculated by DSSP [34] using the pub-lished 3D structures [5,15] for PA83 is H15/S35/T23/O27and PA63 is H15/S30/T27/O28 (percentage of Helix/b-Strand/Turn/Other). Porcine elastase [31], with a very sim-ilar bII CD spectrum [32], has values of H11/S34/T20/O35.The far UV CD data for PA was fitted using the CDSSTRprogram of the Dichroweb site (http://www.cryst.bbk.a-c.uk/cdweb/) [35] using a wavelength range of 190–250 nm,and dataset 4. This gives values for the secondary structurecontents at neutral pH which are very similar to the X-rayderived values. PA83 pH 8.0 (H10/S29/T24/O34), andPA63 pH 8.0 (H13/S30/T24/O33). Surprisingly, althoughthe spectra change from bII to bI at pH 3.0, the secondarystructure contents predicted by CDSSTR are very similarto those at neutral pH PA83 (H13/S31/T23/O33), PA63(H17/S29/T24/O30). This may be because PPII rearrange-ments known to occur in this transition mainly involve rear-rangements within the ‘‘Other’’ category. [32]. Thus the bcontent may not change significantly.

Thermal stability of PA83

Upon heat denaturation the far UV CD spectrumundergoes a shift to a smaller but more canonical bI sheet

Fig. 3. (a) Fluorescence in the presence of PA83 at a range of pH. Intrinsic fluorescence: PA83 (0.61 lM) at various pH was examined at 20 �C withexcitation at 280 nm and emission recorded from 295 to 450 nm. ANS fluorescence: 5.0 lM PA83 was incubated with 60 lM ANS in the presence of50 mM buffer at specified pH for 1 h at 20 �C. The resulting samples were measured at 20 �C with excitation at 370 nm and emission recorded from 400 to600 nm. (Inset) ANS fluorescence: PA83 (0.61 lM) full spectra for pH 4 and pH 9. (b) Far UV-CD spectra of PA83 upon pH induced unfolding. PA83(6 lM) in 50 mM of appropriate pH buffer after overnight incubation (pH 4, 1 h) at 20 �C. Far-UV CD for a range of pH values (2–6, as no change wasobserved at increased pH) in 0.2 mm pathlength cuvette. (Inset) Near UV-CD spectra of 12 lM PA83 incubated at a range of pH overnight at 20 �C. CDspectra were collected in a 1 cm path length cuvette at 20 �C. (c) Far UV CD of PA63 and PA83 (6 lM) in 50 mM of appropriate pH buffer after overnightincubation. CD spectra were collected in a 0.2 mm pathlength cuvette at 20 �C.

6 D.A. Chalton et al. / Archives of Biochemistry and Biophysics 465 (2007) 1–10

Fig. 4. (a) Far UV CD of PA83 in 100 mM phosphate buffer pH 7.0 to obtain short wavelength data which were collected in 0.1 mm quartz cuvette, at ascanning speed of 10 nm/min over 10 accumulations on a JASCO 810 Spectropolarimeter. (b) Far UV CD of PA83 upon heat induced unfolding. PA83(0.2 mg/ml) in 100 mM phosphate buffer pH 7.0, at 25 and 61 �C data were collected in a 1 mm quartz cuvette at a scanning rate of 50 nm/min. (c)Differential Scanning Calorimetry and thermal denaturation measured by Far-UV CD220. DSC analysis of PA83. PA83 at 0.75 mg/ml in 30 mM PIPESwas subjected to DSC with a thermal ramp of 1 �C/min. Heat capacity data are corrected for instrumental (buffer) baseline and concentration normalized.Derived Tm is 49.9 �C. Normalized Far-UV CD220 thermal denaturation profile (1 �C/min) of PA83 (�0.1 mg/ml) in 100 mM phosphate buffer pH 7.0.Derived Tm is 50.5 �C. (A similar Far-UV CD220 thermal denaturation profile in 30 mM PIPES pH 7.0 gave a derived Tm of �49 �C (data not shown).)

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spectrum with a minimum at 215 nm (Fig. 4b). This transi-tion was measured by temperature-gradient far-UV circu-lar dichroism at 220 nm (Fig. 4c) and differentialscanning calorimetry (Fig. 4c). These gave mid-points oftemperature unfolding (Tm) of 49.8 and 47.8 �C, respec-tively. Due to the irreversibility of PA thermal denatur-ation, the raw DSC data is truncated upon theprecipitation of PA (�52 �C) and the calculated enthalpyvalues cannot be fully interpreted. However, the similarityof calorimetric (Hcal = 218 kJ mol�1 (52 kcal mol�1)) andvan’t Hoff (HVH = 235 kJ mol�1 (56 kcal mol�1)) enthalpyvalues do not exclude a simple two-state transition nor isthere evidence for separate domains unfolding at differenttemperatures.

Discussion

The work presented here extends the published data[24,25] by presenting a complete unfolding/refolding profileof PA83, the first published CD spectra of both monomericPA83 and the oligomeric form PA63 and the effect of phys-iological pH changes.

PA83 shows distinct biphasic unfolding and refolding. Acalcium- and pH-dependent destabilization of the C-termi-nal region is the first step in unfolding (and conversely thelast step in refolding). The crystal structure of PA83 [15]

Fig. 5. Left: side view of PA63 monomer taken from PA63 heptamer structurblack space filling representation). Tryptophan residues are shown as space fillin346 is not resolved in the PA83 structure. Domain 1 (PA20) is at the top and coand PA bound calcium ions, respectively.

revealed the presence of two calcium ions in the C-terminalregion (adjacent to domain 1) and the authors proposedthat these ions play an important role in the stabilizationof the PA83 monomer. Gupta et al. [24] show that holo(calcium containing) PA possesses a typical trypsin cleav-age pattern in which PA83 is cleaved to produce a trypsinresistant form, PA63. PA with the stabilizing calcium ionsremoved (Apo-PA), shows sensitivity to protease degrada-tion, and shows no PA63 form remaining. They suggest themaintenance of domain 1 by calcium allows PA63 to olig-omerise and so bind the edema or lethal factor. The initialtransition is thus linked specifically to the stabilizing effectsof calcium ions. This is in agreement with [17] who hypoth-esize that calcium plays a role in stabilizing domain 1 0.Folded Apo-PA83 shows a shift of km to 352 nm fromthe PA83 value of 343 nm. This implies the change thatoccurs with calcium involves a significant rearrangementin a region that contains aromatic residues. The data atpH 4.0 indicate that this localized stabilization by calciumis eliminated in acidic conditions (Fig. 3a).

The refolding data presented here shows clearly that cal-cium is not required for initial PA83 folding since the lackof calcium is only manifested in the inability to bury thetryptophans that form the interface between domain 1and the rest of PA. We also show that a native refoldedPA83 structure can only be achieved from denatured

e (PDB: 1TZO) [4] (insert shows heptamer top view with one monomer asg and numbered. Right: structure of PA83 [15] (PDB: 1ACC). Tryptophanntains residues W65, 90, 136. D2, D3, D4 and Ca indicate domains 2, 3, 4

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PA83 when the refolding buffer contains calcium. Calciumindependent refolding occurs between 1 and 6 M urea,whilst calcium dependent refolding occurs between 0 and1 M urea. This is in agreement with both [24] who showedthat PA lacking calcium has a difference intrinsic fluores-cent spectra, and [17] that calcium added to Apo-PAcaused a return to a more native state.

Fluorescence spectroscopy analyzed with the precisebarycentric mean method is directly relevant to the knownX-ray structure [15] of monomeric PA83 (PDB:1ACC)(Fig. 5). Of the seven tryptophans W346 is not resolved inthe X-ray derived structural model of PA83. The remainderare equally split between those near the calcium binding sites(W447, W226 and W206) and those in the core of domain 1(W65, W90, W136) which is released as PA20 after furincleavage. The biphasic unfolding which is more sharplydefined by fluorescence than CD thus appears to originatefrom an initial destabilization of the domain 1-PA63 inter-face whilst the second fluorescent transition reports ondomain 1 unfolding. The CD spectra which are sensitive tothe entire protein backbone show the initial destabilizationstep and then show that all the domains (including domain1) unfold between 3 and 5 M urea. Thermal unfolding dataalso shows no evidence for significant differences in domainstability and confirms that the biphasic unfolding is due totwo quite different types of structural change.

PA83 shows very little change in fluorescence betweenpH 10 and pH 6 (data not shown, and Fig. 3a) in contrastto previously reported data [25]. However, although a sub-tle change must occur in the structure of PA83 at lower pH,these data suggest that it does not affect the direct environ-ment of aromatic residues since this would involve a notice-able change in the fluorescence data. The CD data showlarge changes affecting both secondary (Fig. 3b) and ter-tiary structure (Fig. 3b inset). Surprisingly, the analysis ofthe far-UV data indicates that the relative amounts of bstructure are the same in both forms. This is possible sincesuch differences may be due to small changes in the amountof the strong PPII signal which separates the bI and bII

spectra classifications of Sreerama and Woody [32]. bI pro-teins display the expected spectrum of a model b sheet witha 215 nm negative maximum whereas bII proteins have theb signal masked by a strong PPII contribution. This is sel-dom seen in helix rich proteins since the spectrum from a-helices is much stronger than b-structure. The structure ofPA is rich in ‘‘other’’ structure as defined by DSSP [34] andhas a very similar spectrum and structural content to a bII

‘‘standard’’ protein porcine elastase [32]. To our knowledgethis is the first example of a thermal or pH driven transitionof a protein from bII to bI spectra form.

Similar pH induced changes are also observed in the CDspectra of the PA63 oligomer, the form which, in nature, isexposed to the low pH of the endosome. The PA63 hepta-mer at low pH in the membrane should contain a fullyformed b-barrel which would explain the changes in CD;however it is the curious similarity of PA83 which is diffi-cult to explain. It suggests that the pH sensitivity is a prop-

erty of PA that exists before oligomerisation althoughin vivo data show that the PA83 monomer is not endocyto-sed and thus not exposed to acidic conditions. Recently,however it has been suggested that uncleaved PA83 mightoligomerise with PA63 to create a (PA83)1(PA63)6 or(PA83)2(PA63)5 hetero-heptamers [26]. Thus the PA83might be able to enter into the same oligomeric associationas PA63 and present the required b hairpin for the pore-structure upon acidification.

In conclusion, we have provided a complete descriptionof the biphasic unfolding and refolding of PA83. We showthat it is initially determined by the destabilization of theinterdomain interactions in the C-terminal region (cal-cium-binding) near domain 1 followed by unfolding of allthe whole domains. We report the first CD data for oligo-meric PA63 and show that it undergoes a bII to bI struc-tural change upon acidification. Furthermore, we showthat PA83 shows the same changes and thus pH dependentrefolding is not restricted to the PA63 heptamer.

Acknowledgments

This research was supported by extramural fundingfrom Dstl (to JHL & DAC) and Wellcome Trust Equip-ment Grants 56232, 40422, 55979. The authors thank An-ton Le Brun for critical reading of the manuscript.

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