Copper(II) binding of prion protein’s octarepeat model peptides

Inorganica Chimica Acta 357 (2004) 185–194

www.elsevier.com/locate/ica

Copper(II) binding of prion protein�s octarepeat model peptides

Giuseppe Pappalardo a,*, Giuseppe Impellizzeri b, Tiziana Campagna a

a Istituto CNR di Biostrutture e Bioimmagini-Sezione di Catania, V.le A. Doria 6, 95125 Catania, Italyb Dipartimento di Scienze Chimiche Universit�aa di Catania, V.le A. Doria 6, 95125 Catania, Italy

Received 29 April 2003; accepted 26 July 2003

Abstract

The complexes between copper(II) and the synthetic octapeptide fragments of the prion protein Ac-GWGQPHGG-NH2 (1), Ac-

PHGGGWGQ-NH2 (3) and the cyclic analogue c-(GWGQPHGG) (2) have been comparatively investigated by circular dichroism

(CD), absorption (UV–Vis), and electron paramagnetic resonance (EPR) spectroscopic methods.

The results suggest a similar copper(II) coordination behaviour of the two linear peptides. In both cases two major complex

species were spectroscopically detected. The first one, existing in the range of pH 7–9, showed spectroscopic parameters attributable

to a 3N complex species, while the 4N complex was the main species at strongly alkaline pH values. Copper(II) binding appears to

be confined within the aminoacid sequence HGG.

Cyclisation of the main chain, as in the peptide 2, was found to have remarkable effects on the copper(II) complex speciation

especially at pH 7–8 where the 3N species predominated in the linear counterparts. By contrast the spectroscopic data obtained at

pH 11 provided evidence of the restoration of the same set of donor atoms as in the linear peptides.

� 2003 Elsevier B.V. All rights reserved.

Keywords: Prion; Copper; Peptides; Metal complexes; Circular dichroism; EPR

1. Introduction

The prion diseases, fatal neurodegenerative disor-

ders, arise from the post-translational conversion of the

normal cellular prion protein (PrPC) into the patho-

logical isoform PrPSc [1]. During this transformationPrPSc becomes infectious, protease-resistant, insoluble

in non-denaturant detergents, and accumulates in af-

fected brains [2]. PrPC and PrPSc differ only in their

secondary and tertiary structures being increased

b-sheet content and decreased a-helices in the patho-

logical isoform PrPSc [1,3]. The mechanism of the

conversion of PrPC into its abnormal isoform PrPSc is

presently unknown, and little is known about the nor-mal function of PrPC in the brain. Very recently PrPC

was shown to be a copper protein [4] and since then a

* Corresponding author. Tel.: +39-095-7385016; fax: +39-095-

337678.

E-mail address: [email protected] (G. Pappalardo).

0020-1693/$ - see front matter � 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0020-1693(03)00492-4

large body of evidence indicated a role for PrPC in

copper(II) metabolism [4–9]. In this respect, several

observations have excited interest in the binding of

copper(II) ions to the PrPC protein as well as in the

structural and functional consequences of such inter-

action. For instance, mice deficient in PrPC showed a>10-fold reduction of copper in a microsomal fraction

from brains relative to wild-type mice and a reduction

in activity of Cu/Zn superoxide dismutase [4a], even if

this has not been replicated by other authors [10].

Moreover, it has been demonstrated that different PrPSc

types, characteristic of clinically distinct subtypes of

sporadic Creutzfeldt–Jakob disease (CJD), can be in-

terconverted in vitro by altering the metal ion occu-pancy [11]. The prion protein has also been proposed to

function as a copper transport protein for internalisa-

tion of copper(II) ions [5,12]. Studies with recombinant

protein and peptides related to its sequence have shown

that prion protein binds copper ions in its N-terminal

region which contains a series of octapeptide repeats

with the consensus sequence PHGGGWGQ [4,13–20],

mail to: [email protected]

186 G. Pappalardo et al. / Inorganica Chimica Acta 357 (2004) 185–194

even if copper(II) binding to the C-terminal domain

and to the region between the repeats and C-terminal

region has been recently reported [21–25].

Solution NMR structures of several recombinant

mammalian PrPC proteins revealed that the N-terminusincluding the octarepeats is highly flexible and unstruc-

tured while the C-terminal region adopts a globular

structure composed of three a-helices and two short b-strands [26–29]. It is reported that copper(II) binding to

the octarepeats not only adds structure in the N-terminal

domain but may also lend stability to the carboxy

terminus [15,19].

We have recently studied the copper(II) complexspecies with the Ac-PHGGGWGQ-NH2 and Ac-

HGGG-NH2 peptides. The results led to the conclusion

that, at neutral pH, metal coordination within an octa-

repeat arises from the His imidazole and amide nitro-

gens encompassing the HGGG peptide fragment [30].

More recently, the solid state structure of the Cu(II)–

HGGGW complex has been reported. It shows, in ad-

dition to the expected involvement of the His imidazoleand the peptide nitrogens, an indirect participation of

the indole nitrogen from the Trp side chain to the co-

ordination of the metal ion [31,32].

To obtain further information on the copper(II)

complex�s structure within the tandem octarepeats, here

we report on the copper(II) complex formation of two

new synthetic peptide models Ac-GWGQPHGG-NH2

(1) and cyclo-(GWGQPHGG) (2).The aim of this study was to investigate, through a

comparative approach, whether the peptides under

study can form copper(II) complexes structurally similar

to those observed for the natural octarepeat peptide

sequence Ac-PHGGGWGQ-NH2 (3) [15,17,18]. This, in

turn, would indicate whether both the polypeptide

chain�s flexibility and primary structure are stringent

parameters to provide the precise copper(II) coordina-tion environment within a single octarepeat.

2. Experimental

2.1. Materials

All N-fluorenylmethyloxycarbonyl(Fmoc)-protectedaminoacids, Novasyn-TGR resin, 2-(1-H-benzotriazole-

1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

(TBTU) and N-hydroxybenzotriazole (HOBT), were

purchased from Novabiochem (Switzerland). N,N-

Dimethylformamide (DMF, peptide synthesis grade)

and 20% piperidine/DMF solution, were from Applied

Biosearch. N,N-Diisopropylethyl amine (DIEA), triiso-

propylsilane (TIS), ethanedithiol (EDT), thioanisole andtrifluoroacetic acid (TFA) were from Sigma/Aldrich. All

other chemicals were of the highest available grade and

were used without further purification.

2.2. Peptide synthesis

The synthesis and purification of Ac-PHGGGWGQ-

NH2 (3) has been reported elsewhere [30]. The peptide

Ac-GWGQPHGG-NH2 (1) was synthesised by solidphase peptide synthesis (SPPS) on a Milligen 9050

continuous flow peptide synthesizer and employing

Fmoc (9-fluorenylmethyloxycarbonyl) chemistry. The

glycine residue was linked to the Novasyn-TGR resin

(Novabiochem). In both the linear peptides, the C-

terminal carboxylate group was synthesised in the

amide form, while the N-terminal amino group was

acetylated with 0.3 M Ac2O in DMF after completionof the synthesis. This was done to suppress participa-

tion of the free amino group in metal complexation and

to better reproduce the native sequence in the parent

protein.

The cyclo-(GWGQPHGG) (2) was obtained by cyc-

lisation of the linear peptide precursor 2HN-GW

GQPHGG-OH, which was assembled as above by SPPS

starting from Fmoc-Gly-PepSyn-Ka resin (Milligen).After completion of the synthesis, both the linear pre-

cursor and peptide 1 were cleaved off from the respective

resins by treatment with a mixture of TFA/phenol/H2O/

EDT/TIS/thioanisole (82.5/5.0/5.0/2.5/2.5/2.5 v/v/v/v/v/

v) for 1.5 h. The linear peptide 2HN-GWGQPHGG-OH

was cyclised in DMF solution under high dilution and in

the presence of TBTU/HOBT/DIEA.

Purification of the peptides was carried out by semi-preparative reversed-phase HPLC on a 250� 10 mm

Vydac C18 (5 lm particle size, 300-�AA pores). Each pep-

tide was eluted isocratically with 12% acetonitrile/water/

0.1% TFA at a flow rate of 3 ml/min. Elution profiles

were monitored at 278 nm. The products were charac-

terised by 1H NMR spectroscopy and FAB-MS spec-

trometry [peptide 1: m=z 836.4 (M+H)þ calc. for

C37H49N13O10 835.37; peptide 2: 777.2 (M+H)þ calc.for C35H44N12O9 776.33].

2.3. Spectroscopic measurements

2.3.1. Circular dichroism (CD)

The CD spectra were obtained at 25 �C under a

constant flow of nitrogen on a Jasco model J-810

spectropolarimeter which had been calibrated with anaqueous solution of ammonium DD-camphorsulfate

[33]. Experimental measurements were carried out in

water and at different pH values using a 1-mm or

1-cm path length cuvette. The CD spectra pertinent to

the free peptide ligands were recorded in the UV re-

gion (190–260 nm), whereas those in the presence

of Cu2þ were examined in the wavelength range of

190–300 and 300–800 nm. The spectra represent theaverage of 8–20 scans. CD intensities are expressed in

De (M�1 cm�1).

-10

0

10

190 200 210 220 230 240 250 260

1

2

3

(M-1

cm

-1)

Wavelength (nm)

-10

0

10

190 200 210 220 230 240 250 260

1

2

3

∆ε(M

-1 c

m-1

)

Wavelength (nm)

Fig. 1. CD spectra of Ac-GWGQPHGG-NH2 (1), C-(GWGQPHGG)

(2) and Ac-PHGGGWGQ-NH2 (3) in H2O, pH 7.

G. Pappalardo et al. / Inorganica Chimica Acta 357 (2004) 185–194 187

2.3.2. NMR spectroscopy

Five millimolar solution samples were prepared in 90/

10 H2O/D2O or pure D2O containing trimethylsilyl-

propionic acid (TSP) as the internal standard. The pH of

the solution was adjusted to the desired value by addingthe suitable acid or base solution. The measured elec-

trode pH values are uncorrected for the isotope effect.

All NMR spectra were acquired at room temperature on

a Varian INOVA Unity-plus spectrometer operating at

499.884 MHz. One-dimensional spectra were normally

acquired with 32K data points over a spectral width of

6000 Hz. Two-dimensional experiments were typically

acquired with 2K data points in the t2 dimension and512 t1 increments. Water saturation was achieved by low

power irradiation during the relaxation delay. ROESY

and NOESY spectra were run at mixing times of 300

and 500 ms, respectively, whereas the TOCSY experi-

ments were acquired using a 80 ms mixing time.

2.3.3. ESR spectroscopy

Frozen solution ESR spectra were recorded on aBruker ER 200 D spectrometer equipped with the 3220

data system at 150 K. Copper(II) complex solutions

were prepared in situ by mixing the necessary volume of

a standard solution of 63Cu(NO3)2 with solutions of the

peptide ligand in 1:1 metal to ligand ratio, and adjusting

the pH of the resulting solution to the desired value by

adding 10 mmol/dm3 KOH or HNO3. Methanol or

trifluoroethanol (TFE) not exceeding 10% was added tothe aqueous copper(II) complex solutions to increase

resolution.

3. Results and discussion

3.1. Metal-free ligands

The first step of our study concerns the evaluation of

the conformational preferences of the peptides in

aqueous solution by means of CD and NMR spectros-

copies. Fig. 1 shows the CD spectra of peptides 1 and 2

in aqueous solution and at pH 7.4. For comparison the

CD spectrum of the octarepeat Ac-PHGGGWGQ-NH2

(3) [30] in the same experimental conditions is also re-

ported. Both peptides 1 and 3 display nearly the sameCD curves with a strong minimum around 200 nm and a

small positive signal at around 225 nm. A similar CD

pattern has been also observed in a longer peptide

fragment containing the multiple consecutive octare-

peats and it has been associated with predominantly

unstructured peptide-chain conformation [34]. The

conformationally constrained cyclic analogue 2 shows a

different CD spectrum which consists of positive ellip-ticity at 200 nm along with smaller positive and negative

bands at 220 and 232 nm, respectively (Fig. 1). It is

known that cyclic peptides can provide conformation-

ally well-defined models of turn conformations of the

peptide chain [35]. In the present case, the negative el-

lipticity at 232 nm and the positive band around 200 nm

might be related with the presence of a b turn confor-

mation [34]. Analogous to the CD spectra of 1 and 3, the

positive signal around 220 nm, observed in 2, can be

assigned to an aromatic transition of the tryptophan

side chain [36].Variations of the pH of the solution did not appre-

ciably affect the overall shape of the CD curves of all the

peptides here studied, thus suggesting that little or none

conformational change is induced by the pH (not

shown).

One-dimensional and two-dimensional NMR exper-

iments were recorded in aqueous solution at pH 4.

Resonance assignments were achieved using g-COSY[37] and TOCSY [38] spectra and then sequential as-

signments were extracted by inspection of the dipolar

connectivities in the NOESY [39] or ROESY [40] spec-

tra. It should be said that in all the peptides here in-

vestigated the cis/trans isomerism around the Gln-Pro

peptide bond in peptides 1 and 2 and around the Ac-Pro

bond in peptide 3 has been observed and the assign-

ments reported in Table 1 are relevant to the predomi-nant trans isomer.

As regards the solution conformation of the linear

peptides 1 and 3 the information derived from the NMR

experiments are consistent with those obtained from CD

experiments: the observation of only appreciably intense

sequential daN NOE connectivities suggests that the

peptides essentially adopt an extended and flexible

conformation in solution [41]. Moreover, the resultsobtained for peptide 3 are similar to other previously

reported NMR studies [32]. This is not surprising if we

consider that the glycine residues amount to 50% of the

entire amino acid sequence. In this regard it has been

hypothesised that the glycine residues may play an

important role in maintaining the necessary peptide

Table 1

Resonance assignments for the linear (1 and 3) and cyclic (2) peptides

Residue NH Ha Hb Others

Peptide 1 Ac-GWGQPHGG-NH2

Ac 1.93

Gly 8.14 3.82

Trp 8.01 4.62 3.31, 3.26 2H 7.25; 4H 7.62; 5H 7.17; 6H 7.25; 7H 7.50; NH 10.14

Gly 8.33 3.80

Gln 8.01 4.58 2.06, 1.89 cCH2 2.32; NH2 7.59, 6.87

Pro 4.38 2.26, 1.84 cCH2 1.99; dCH2 3.76, 3.64

His 8.68 4.65 3.27, 3.18 2H 8.57; 4H 7.31

Gly 8.47 3.97

Gly 8.32 3.92

NH2 7.53; 7.08

Peptide 2 cyclo-(GWGQPHGG)

Gly 8.06 3.94

Trp 8.61 4.39 3.32 2H 7.28; 4H 7.64; 5H 7.20; 6H 7.27; 7H 7.54; NH 10.12

Gly 8.48 3.80, 3.42

Gln 7.65 4.65 2.16, 1.84 cCH2 2.32; NH2 7.48, 6.85

Pro 4.36 2.22, 1.63

His 8.50 4.68 3.40, 3.26 2H 8.61; 4H 7.34

Glya 8.28 4.21, 3.90

Glya 8.02 3.99

Peptide 3 Ac-PHGGGWGQ-NH2

Ac 2.04

Pro 4.27 2.16; 1.90

His 8.59 4.59 3.21, 3.12; 2H 8.49, 4H 7.19

Gly 8.39 4.01

Glya 8.32 3.92

Glya 8.32 3.92

Trp 8.09 4.43 3.31; 3.24 2H 7.23; 4H 7.61; 5H 7.16; 6H 7.23; 7H 7.48; NH 10.11

Gly 8.32 3.84, 3.76

Gln 8.05 4.27 2.10; 1.91 cCH2 2.28; NH2 7.50, 6.83

NH2 7.61; 7.09a Interchangeable values.


chain flexibility in the prion�s protein N-terminal region

to provide a very specific coordination environment for

the Cu(II) ions [42].

The glycine residues are expected to allow a certaindegree of backboneflexibility also in the cyclic analogue 2;

indeed, the measured 3JNH–aH coupling constants of the

glycine residues are near the conformationally averaged

value of 6–7 Hz [43]. In addition, the NMR analysis did

not reveal sufficient transannular NOEs to permit the

precise conformation. However, the presence of NOE

cross-peaks between the NH protons of Gly3 and Gln4dNN (i; iþ 1) and between the C–Ha of Trp2 and the N–Hof Gly3 protons daN (i; iþ 1) (not shown), together with

the significative upfield shift of the Trp2 and Gly3 C–Ha

protons, may support the hypothesis of the presence of

turn conformation involving the GlyTrpGlyGln residues

[43]. In this respect larger deviations from the random coil

chemical shift values were detected only for the N–H and

C–Ha protons of the cyclopeptide 2 (Fig. 2).

3.2. Copper(II) complexes

CD spectroscopy can provide useful information on

the structural characterisation of metal–peptide com-

plexes as it is very sensitive to ligand coordination ge-

ometry around the metal centre [45]. The visible region

CD spectra of the copper(II)–1 complex recorded at

different pH values are shown in Fig. 3(a). All the CDcurves show two opposite signed bands due to the d–d

electronic transitions [45]. Another positive signal is

observable around 315–335 nm and can be assigned to

optically active ligand to metal charge-transfer transi-

tions (LMCT) that may occur from imidazole or de-

protonated peptide nitrogens to the copper ion [46].

These induced dichroic bands are clearly visible starting

from pH 6.0, thus suggesting the anchoring function ofthe histidine residue in the co-ordination of the metal

ion.

Interestingly, the Cu(II) complex of the single octa-

repeat peptide 3 has the same sign CD bands as the

copper(II)–1 complex in the whole pH range investi-

gated in this study (Fig. 3(b)). The only apparent dif-

ferences are in the intensities of the two series of spectra

with those of the copper(II)–3 complex being more in-tense up to pH 9 while in very strong alkaline conditions

the situation appears overturned. Luczkowski et al. in a

recent potentiometric and spectroscopic study, carried

out on the Cu(II)–Ac-PHGGGWGQ-NH2 complex,

Fig. 2. Comparative plot of the CHa (a) and NH (b) chemical shift deviation from the random coil value (Dd ¼ dobs � drandom coil) of the peptides Ac-

GWGQPHGG-NH2 ( ), c-(GWGQPHGG) ( ), and Ac-PHGGGWGQ-NH2( ) in 90% H2O/10% D2O. Random coil values were taken from

Wishart et al. [44] Note that the plotted values relative to the Ac-PHGGGWGQ-NH2 peptide are irrespective of their position along the sequence.

-0.8-0.6-0.4-0.20.00.20.40.60.81.01.2

300 400 500 600 700 800-0.8-0.6-0.4-0.20.00.20.40.60.81.01.2

300 400 500 600 700 800Wavelength

(M-1

cm-1

)

pH 6

pH 7pH 8

pH 9

pH 10pH 11

pH 6

pH 7; pH 8

pH 9

pH 10 pH 11

-0.8-0.6-0.4-0.20.00.20.40.60.8

1.2

300 400 500 600 700 800-0.8-0.6-0.4-0.20.00.20.40.6

300 400 500 600 700 800-0.8-0.6-0.4-0.20.00.20.40.60.81.01.2

300 400 500 700 800--0.6-0.4-0.20.00.20.40.60.81.01.2

300 400 500 700

∆ε(M

-1cm

-1)

pH 6

pH 7pH 8

pH 9

pH 10pH 11

pH 6

pH 7; pH 8

pH 9

pH 10 pH 11

(a) (b)

Fig. 3. Visible region CD spectra of Cu(II) complexes with 1 (panel a) and 3 (panel b) at different pH values (indicated on the curves).


G

2600 2800 3000 3200 3400 3600G

2

31

2600 2800 3000 3200 3400 3600

2

3

1

2600 2800 3000 3200 3400 3600

2

31

2600 2800 3000 3400 3600

2

3

1

(b)

(a)

Fig. 4. Selected X-band EPR spectra. (a) peptide 1 at pH 8; peptide 3 at

pH 7 and peptide 2 at pH 7; (b) peptide 1, peptide 3 and peptide 2 at

pH 11.


stated that in the range of pH 6–8 the major species is

the CuH-2L complex which involves the imidazole ni-

trogen and two amide nitrogens from the two sub-

sequent glycine residues. Such a complex species gave a

CD spectrum with the characteristic CD bands due toelectronic d–d transition at 727 and 601 nm, together

with the CT transition at 338 nm [32].

In addition, the CD spectra of the copper(II)–1 and

copper(II)–3 peptide systems show a CD pattern that is

rather similar to that observed for themultiple octarepeat

peptides in the samewavelength region [15,17]. Therefore,

it might be likely that also in the copper(II)–1 complex the

same set of donor atoms is retained and difference in thecoordination geometry relative to single and multiple re-

peat peptides is negligible. Further support for this comes

fromEPR andUV–Vis absorption data (Fig. 4 and Table

2): very similar magnetic parameters have been obtained

at neutral pH for both the copper(II) complexes with the

peptides 1 and 3; moreover, these values are not distant

from those reported either for single or multiple octare-

peat copper(II) peptide complexes at physiologic pH[18,22,30]. The measured values obtained at pH 7–8 are

consistent with those of an in-plane 3N complex species

and the presence of the CD bands at 333 and 337 nm for

the copper(II)–1 and –3 complexes, respectively, indicates

the involvement of the imidazole in the metal ion binding

[46]. On the other hand, the comparison of the electronic

spectra absorption values in the range of pH 6–8 (Table 2)

suggests that in the case of the copper(II)–3 system theformation of the 3N complex species occurs at lower pH

values.

Above pH 8 the CD spectra of the two systems

gradually undergo distinct changes mainly in the d–d

region, while the CT band moves toward shorter

wavelength and decreases in intensity (Fig. 3).

Apart from their intensity, the pH 11 CD spectra of

both peptide complexes reveal nearly identical features,thus providing further evidence for the presence of quite

similar chromophores. Again, this observation is nicely

confirmed by the UV–Vis absorption values and EPR

parameters that are almost coincident in both systems

and are characteristic of a 4N complex species (Fig. 4

and Table 3). In agreement with recent data that ap-

peared in the literature [47], we hypothesise that in such

complex species also the amide nitrogen of the histidineresidue is engaged in copper(II) co-ordination to afford

a {NIm; 3N�} set of donor atoms in the equatorial

coordination plane.

In addition, the comparison of our CD results with

the literature data relevant to copper(II) complexes with

the HGG, HGGG and HGGGW peptides [17,48] al-

lowed the conclusion that the differences seen in the CD

intensities for the two copper(II)–1 and copper(II)–3complexes may be correlated with the proximity of the

Trp side chain to the copper(II) coordination site in the

copper(II)–3 system.

CD spectra in the UV region have been recorded toestablish whether the addition of Cu2þ to peptide 1

causes structuring of the peptide backbone from the

unstructured conformation as indicated above by the

CD spectrum shown in Fig. 1. The influence of cop-

per(II) on the main chain conformation of the octare-

peat peptide 3 has been previously reported [30].

The CD spectra collected in the range of pH 5–11

show an influence of the copper(II) on the solutionconformation of 1 starting from pH >6 (Fig. 5). This

time however, the CD curves profoundly differ when

compared with those observed for the parent peptide

analogue 3 [30]. For instance, the distinctive negative

ellipticity at 216 nm, suggestive of structure formation in

the CD spectra of the copper(II)–3 complex, is never

observed in the case of the copper(II) complex with 1;

Table 2

Spectroscopic data for the copper(II) complexes of Ac-GWGQPHGG-NH2 and Ac-PHGGGWGQ-NH2a

pH UV–Vis CD EPR

k (nm) e (M�1 cm�1) k (nm) De (M�1 cm�1) Ak gk

5.0 800 [790] 13 [16]

6.0 700 [645] 18 [26] 333 [338]c 0.042 [0.170] b [149] b [2.348]

588 [598]d 0.036 [0.212]

693 [727]d )0.021 [)0.094]

7.0 634 [625] 50 [87] 333 [337]c 0.257 [0.641] 167 [165] 2.230 [2.236]

584 [598]d 0.184 [0.815]

698 [717]d )0.162 [)0.331]

8.0 625 [622] 61 [87] 333 [337]c 0.395 [0.682] 165 [164] 2.231 [2.237]

584 [598]d 0.268 [0.817]

697 [717]d )0.294 [)0.336]

9.0 605 [605] 62 [79] 332 [335]c 0.390 [0.570] 166 [163] 2.232 [2.235]

500d )0.062588 [598]d 0.295 [0.560]

697 [717]d )0.210 [)0.256]

10.0 550 [545] 92 [103] 322 [332]c ;e 0.280 [0.394] 166 [b] 2.230 [b]

508 [498]d )0.254 [)0.181]593 [584]d 0.270 [0.159]

702d )0.067

11.0 538 [535] 121 [134] 315 [327]c ;e 0.307 [0.273] 201 [201] 2.189 [2.193]

490 [494]d )0.607 [)0.665]571 [574]d 0.960 [0.481]

a Values for the Ac-PHGGGWGQ-NH2 are given in brackets.b Parameters not obtained.cNim !Cu2þ charge transfer.d d–d transition.eN� !Cu2þ charge transfer.


instead it shows the persistence of negative ellipticity

below 200 nm even at strongly alkaline pH values. By

contrast, at these high values of pH, the CD spectra of

the copper(II)–3 complex were characterised by dimin-

ished negative ellipticity at 216 nm and positive signal at

ca. 203 nm [30]. Paradoxically, a part of greater ellip-

ticity at 226 nm, the pH 11 CD spectrum of the cop-

per(II)–1 complex, resembles that of the free peptideligand. The conclusion that emerges from these data is

that the sequence of amino acids required to observe the

conformational effects within the single octarepeat

peptide 3 appears quite specific.

As far as the cyclic analogue 2 is considered, signifi-

cant differences in the CD, EPR and UV–Vis parameters

are observed (Table 3). The CD spectra recorded in the

300–800 nm wavelength range show double signed d–dbands, typical of Cu(II)–His engagement [45], starting

from pH 7. These bands gradually increase in intensity

and shift toward shorter wavelength by the increasing

pH while the growth of a positive CT band at 314–326

nm becomes detectable from pH 9 onward (Fig. 6).

The CD spectra collected at pH 7 and pH 8 appear of

weak intensity also displaying a different pattern with

respect to those of linear analogues 1 and 3 at the samepH values. Furthermore, no appreciable ellipticity was

detected in the range of 300–350 nm. These data might

argue for the presence of complex species with distinctly

different coordination geometry. EPR spectra recorded

in the range of pH 7–9 exhibited signals in the gk regionthat were interpreted in terms of simultaneous presence

of copper (II) complexes having different magnetic pa-

rameters (Fig. 4). Unfortunately, the spectroscopic data

do not allow the structural characterisation of thecomplex species. However, the absence of the typical

midfield transition together with experiments recorded

at various metal to ligand ratios ruled out the presence

of dimeric species [49]. What is apparent is that, at these

pH values, the coordination mode in the copper(II)

complex of the peptide 2 appears to be different from

the linear peptide counterparts 1 and 3. It is likely that

the reduced conformational mobility of the cyclopep-tide 2 could be responsible for such a different behav-

iour toward copper(II) binding. On the other hand, at

pH 11 EPR signals attributable to a single major com-

plex species are observed (Fig. 4). The measured mag-

netic parameters are consistent with a complex having

four co-ordinated nitrogens in the equatorial plane

(Table 3). Interestingly, the CD spectrum recorded at

this pH nearly reproduces, in shape and intensity, thepattern observed for the copper(II)–1 complex at the

Table 3

Spectroscopic data for the copper(II) complexes of cyclo(GWGQPHGG)

pH UV–Vis CD EPR

k (nm) e (M�1 cm�1) k (nm) De (M�1 cm�1) Ak gk

5.0 780 17

6.0 707 29

7.0 656 68 349b )0.027 a a

590c )0.062693c 0.036

8.0 630 85 347b )0.046 a a

590c )0.100693c 0.095

9.0 570 91 324b ;d 0.100 a a

516c )0.120595c )0.061700c 0.100

10.0 546 115 326b ;d 0.362 a a

507c )0.334592c 0.143

11.0 552 132 318b ;d 0.603 199 2.190

496c )0.705584c 0.891

a Parameters not obtained.bNim !Cu2þ charge transfer.c d–d transition.dN� !Cu2þ charge transfer.

-16

-12

-8

-4

0

4

8

12

190 200 210 220 230 240 250 260

pH 11

pH 10pH 5

pH 6

pH 7pH 8

pH 9

Wavelength

∆ε(M

-1cm

-1)

Fig. 5. CD spectra of the copper(II)–1 complex in H2O at different pH

values (indicated on the curves).

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

300 400 500 600 700 800

Wavelength

∆ε(M

-1cm

-1) pH 7

pH 5

pH 6pH 8

pH 11 pH 10

pH 9

Fig. 6. Visible region CD spectra of the Copper(II)-2 complex with at

different pH values (indicated on the curves).


corresponding pH value. Therefore, it argues that the

nature of the donor centres could be the same as the

linear analogues.

Looking at the CD spectra recorded in the UV re-

gion, (Fig. 7) it is evident that major effects on the cy-clopeptide�s solution conformation occur above pH 8.

Interestingly, these changes parallel the growth of the

band at 314–326 nm and suggest that the major one

responsible for the conformational changes is the com-

plex species with deprotonated peptide nitrogen engaged

in copper(II) binding.

4. Conclusion

In the present study the copper(II) coordinationproperties of three prion peptide fragments have been

comparatively investigated.

-10

0

10

190 200 210 220 230 240 250 260

Wavelength (nm)

∆ε(M

-1 c

m-1

)

Fig. 7. CD spectra of the copper(II)–2 complex in H2O at different pH

values. By following the direction of the arrows pH 5.0; pH 6.0; pH

7.0; pH 8.0; pH 9.0; pH 10.0; pH 11.0.


The results obtained indicate that despite the different

primary structures, the two linear peptides 1 and 3 be-

have similarly toward copper(II) complexation. From

the spectroscopic data only two major complex species

can be identified for the copper(II)–1 system: a 3Ncomplex, occurring in the pH range 7–9, which involves

the imidazole nitrogen of the histidine residue and the

two peptide nitrogens from the subsequent C-terminal

glycines and the 4N type complex, that predominates at

pH 11, in which also the amide nitrogen of the histidine

residue probably enters into the copper(II) co-ordina-

tion sphere. Likewise, for the copper(II)–3 system the

same set of donor atoms can be identified within theHGG residues in the middle of the peptide sequence. In

this case however an additional complex species can be

hypothesised to occur at about pH 6.

The analysis of the CD experiments recorded for the

cyclopeptide 2 indicates that copper(II) binds to cyclo-

peptide 2 around pH 7 presumably through the histi-

dine�s imidazole side chain. However, the complex

species formed around the neutrality and slightly basicpH values distinctly differ from those of the parent lin-

ear peptides.

Overall these data show that either primary sequence

or main chain flexibility appears to be necessary to

provide the precise metal binding environment and the

specific folding within a single octarepeat.

Acknowledgements

We acknowledge CNR Agenzia 2000 project

CNRC00781B, MIUR FIRB 2001 project RBNE01A-

RRAandMIURPRIN 2001 Project 2001031717_003 for

financial support. Thanks are also due to Prof. Enrico

Rizzarelli for helpful discussions.

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Copper(II) binding of prion protein’s octarepeat model peptides

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Transcript of Copper(II) binding of prion protein’s octarepeat model peptides