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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 proteinÕs octarepeat model peptides Giuseppe Pappalardo a
a,*
, Giuseppe Impellizzeri b, Tiziana Campagna
a
Istituto CNR di Biostrutture e Bioimmagini-Sezione di Catania, V.le A. Doria 6, 95125 Catania, Italy b Dipartimento di Scienze Chimiche Universit a 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), AcPHGGGWGQ-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. Ó 2003 Elsevier B.V. All rights reserved. Keywords: Prion; Copper; Peptides; Metal complexes; Circular dichroism; EPR
1. Introduction The prion diseases, fatal neurodegenerative disorders, arise from the post-translational conversion of the normal cellular prion protein (PrPC ) into the pathological isoform PrPSc [1]. During this transformation PrPSc becomes infectious, protease-resistant, insoluble in non-denaturant detergents, and accumulates in affected brains [2]. PrPC and PrPSc differ only in their secondary and tertiary structures being increased b-sheet content and decreased a-helices in the pathological 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 normal 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: +39-095337678. E-mail address: [email protected] (G. Pappalardo). 0020-1693/$ - see front matter Ó 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 interaction. 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 interconverted in vitro by altering the metal ion occupancy [11]. The prion protein has also been proposed to function as a copper transport protein for internalisation 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],
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2.2. Peptide synthesis
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-terminus including the octarepeats is highly flexible and unstructured while the C-terminal region adopts a globular structure composed of three a-helices and two short bstrands [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) complex species with the Ac-PHGGGWGQ-NH2 and AcHGGG-NH2 peptides. The results led to the conclusion that, at neutral pH, metal coordination within an octarepeat arises from the His imidazole and amide nitrogens encompassing the HGGG peptide fragment [30]. More recently, the solid state structure of the Cu(II)– HGGGW complex has been reported. It shows, in addition to the expected involvement of the His imidazole and the peptide nitrogens, an indirect participation of the indole nitrogen from the Trp side chain to the coordination of the metal ion [31,32]. To obtain further information on the copper(II) complexÕs 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 chainÕs flexibility and primary structure are stringent parameters to provide the precise copper(II) coordination environment within a single octarepeat.
The synthesis and purification of Ac-PHGGGWGQNH2 (3) has been reported elsewhere [30]. The peptide Ac-GWGQPHGG-NH2 (1) was synthesised by solid phase 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 Cterminal carboxylate group was synthesised in the amide form, while the N-terminal amino group was acetylated with 0.3 M Ac2 O in DMF after completion of the synthesis. This was done to suppress participation 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 cyclisation 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 precursor and peptide 1 were cleaved off from the respective resins by treatment with a mixture of TFA/phenol/H2 O/ 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 semipreparative reversed-phase HPLC on a 250 10 mm pores). Each pepVydac C18 (5 lm particle size, 300-A 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 characterised by 1 H NMR spectroscopy and FAB-MS spectrometry [peptide 1: m=z 836.4 (M + H)þ calc. for C37 H49 N13 O10 835.37; peptide 2: 777.2 (M + H)þ calc. for C35 H44 N12 O9 776.33].
2. Experimental
2.3. Spectroscopic measurements
2.1. Materials
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 an aqueous solution of ammonium D -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 region (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 the average of 8–20 scans. CD intensities are expressed in De (M1 cm1 ).
All N-fluorenylmethyloxycarbonyl(Fmoc)-protected aminoacids, Novasyn-TGR resin, 2-(1-H-benzotriazole1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and N-hydroxybenzotriazole (HOBT), were purchased from Novabiochem (Switzerland). N,NDimethylformamide (DMF, peptide synthesis grade) and 20% piperidine/DMF solution, were from Applied Biosearch. N,N-Diisopropylethyl amine (DIEA), triisopropylsilane (TIS), ethanedithiol (EDT), thioanisole and trifluoroacetic acid (TFA) were from Sigma/Aldrich. All other chemicals were of the highest available grade and were used without further purification.
G. Pappalardo et al. / Inorganica Chimica Acta 357 (2004) 185–194
2.3.3. ESR spectroscopy Frozen solution ESR spectra were recorded on a Bruker 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 63 Cu(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 to the 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 spectroscopies. 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 reported. Both peptides 1 and 3 display nearly the same CD 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 octarepeats 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 ellipticity 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-
10
∆ε (M-1 cm-1 )
2.3.2. NMR spectroscopy Five millimolar solution samples were prepared in 90/ 10 H2 O/D2 O or pure D2 O containing trimethylsilylpropionic acid (TSP) as the internal standard. The pH of the solution was adjusted to the desired value by adding the suitable acid or base solution. The measured electrode 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 and 512 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 experiments were acquired using a 80 ms mixing time.
187
2
0
3 1
-10
190
200
210
220 230 240 Wavelength (nm)
250
260
Fig. 1. CD spectra of Ac-GWGQPHGG-NH2 (1), C-(GWGQPHGG) (2) and Ac-PHGGGWGQ-NH2 (3) in H2 O, pH 7.
ally well-defined models of turn conformations of the peptide chain [35]. In the present case, the negative ellipticity at 232 nm and the positive band around 200 nm might be related with the presence of a b turn conformation [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 appreciably 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 experiments were recorded in aqueous solution at pH 4. Resonance assignments were achieved using g-COSY [37] and TOCSY [38] spectra and then sequential assignments were extracted by inspection of the dipolar connectivities in the NOESY [39] or ROESY [40] spectra. It should be said that in all the peptides here investigated 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 assignments reported in Table 1 are relevant to the predominant 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 results obtained 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
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Table 1 Resonance assignments for the linear (1 and 3) and cyclic (2) peptides Residue
NH
Peptide 1 Ac-GWGQPHGG-NH2 Ac Gly 8.14 Trp 8.01 Gly 8.33 Gln 8.01 Pro His 8.68 Gly 8.47 Gly 8.32 NH2 Peptide 2 cyclo-(GWGQPHGG) Gly 8.06 Trp 8.61 Gly 8.48 Gln 7.65 Pro His 8.50 Glya 8.28 Glya 8.02 Peptide 3 Ac-PHGGGWGQ-NH2 Ac Pro His 8.59 Gly 8.39 Glya 8.32 Glya 8.32 Trp 8.09 Gly 8.32 Gln 8.05 NH2 a
Ha
Hb
Others 1.93
3.82 4.62 3.80 4.58 4.38 4.65 3.97 3.92
3.31, 3.26
2H 7.25; 4H 7.62; 5H 7.17; 6H 7.25; 7H 7.50; NH 10.14
2.06, 1.89 2.26, 1.84 3.27, 3.18
cCH2 2.32; NH2 7.59, 6.87 cCH2 1.99; dCH2 3.76, 3.64 2H 8.57; 4H 7.31
7.53; 7.08 3.94 4.39 3.80, 3.42 4.65 4.36 4.68 4.21, 3.90 3.99
3.32
2H 7.28; 4H 7.64; 5H 7.20; 6H 7.27; 7H 7.54; NH 10.12
2.16, 1.84 2.22, 1.63 3.40, 3.26
cCH2 2.32; NH2 7.48, 6.85 2H 8.61; 4H 7.34
2.04 4.27 4.59 4.01 3.92 3.92 4.43 3.84, 3.76 4.27
2.16; 1.90 3.21, 3.12;
2H 8.49, 4H 7.19
3.31; 3.24
2H 7.23; 4H 7.61; 5H 7.16; 6H 7.23; 7H 7.48; NH 10.11
2.10; 1.91
cCH2 2.28; NH2 7.50, 6.83 7.61; 7.09
Interchangeable values.
chain flexibility in the prionÕs 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 certain degree of backbone flexibility also in the cyclic analogue 2; indeed, the measured 3 JNH–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 Gln4 dNN (i; i þ 1) and between the C–Ha of Trp2 and the N–H of Gly3 protons daN (i; i þ 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 geometry 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 CD curves 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 transitions (LMCT) that may occur from imidazole or deprotonated peptide nitrogens to the copper ion [46]. These induced dichroic bands are clearly visible starting from pH 6.0, thus suggesting the anchoring function of the histidine residue in the co-ordination of the metal ion. Interestingly, the Cu(II) complex of the single octarepeat peptide 3 has the same sign CD bands as the copper(II)–1 complex in the whole pH range investigated in this study (Fig. 3(b)). The only apparent differences are in the intensities of the two series of spectra with those of the copper(II)–3 complex being more intense 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,
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189
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 AcGWGQPHGG-NH2 ( ), c-(GWGQPHGG) ( ), and Ac-PHGGGWGQ-NH2 ( ) in 90% H2 O/10% D2 O. 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.
1.2 (a)
1.0 0.8 0.4
0.6
pH 7
pH 8 pH 9
0.2
0.2 0.0
-0.2
-0.2
pH 6 pH 10
-0.4 pH 11
-0.6 300
400
pH 9
pH 6
0.4
0.0
-0.8
(b)
pH 7; pH 8
1.0 0.8
0.6
∆ε (M-1 cm-1)
1.2
pH 10 pH 11
-0.4 -0.6
500
600
700
800
--0.8
300
400
500
600
700
800
Wavelength 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).
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stated that in the range of pH 6–8 the major species is the CuH-2 L complex which involves the imidazole nitrogen and two amide nitrogens from the two subsequent glycine residues. Such a complex species gave a CD spectrum with the characteristic CD bands due to electronic 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 the multiple octarepeat peptides in the same wavelength 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 the coordination geometry relative to single and multiple repeat peptides is negligible. Further support for this comes from EPR and UV–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 octarepeat 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 the formation 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 appeared in the literature [47], we hypothesise that in such complex species also the amide nitrogen of the histidine residue 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] allowed the conclusion that the differences seen in the CD intensities for the two copper(II)–1 and copper(II)–3 complexes may be correlated with the proximity of the Trp side chain to the copper(II) coordination site in the copper(II)–3 system.
(a)
1 3 2
2600
2800
3000
3200
3400
3600
G (b)
1 3 2
2600
2800
3000
3200
3400
3600
G 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.
CD spectra in the UV region have been recorded to establish 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 copper(II) on the main chain conformation of the octarepeat 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 solution conformation 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;
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191
Table 2 Spectroscopic data for the copper(II) complexes of Ac-GWGQPHGG-NH2 and Ac-PHGGGWGQ-NH2 a pH
UV–Vis
CD
k (nm)
e (M1 cm1 )
5.0
800 [790]
13 [16]
6.0
700 [645]
7.0
EPR
k (nm)
De (M1 cm1 )
Ak
gk
18 [26]
333 [338]c 588 [598]d 693 [727]d
0.042 [0.170] 0.036 [0.212] )0.021 [)0.094]
b
b
634 [625]
50 [87]
333 [337]c 584 [598]d 698 [717]d
0.257 [0.641] 0.184 [0.815] )0.162 [)0.331]
167 [165]
2.230 [2.236]
8.0
625 [622]
61 [87]
333 [337]c 584 [598]d 697 [717]d
0.395 [0.682] 0.268 [0.817] )0.294 [)0.336]
165 [164]
2.231 [2.237]
9.0
605 [605]
62 [79]
332 [335]c 500d 588 [598]d 697 [717]d
0.390 [0.570] )0.062 0.295 [0.560] )0.210 [)0.256]
166 [163]
2.232 [2.235]
10.0
550 [545]
92 [103]
322 [332]c;e 508 [498]d 593 [584]d 702d
0.280 [0.394] )0.254 [)0.181] 0.270 [0.159] )0.067
166 [b ]
2.230 [b ]
11.0
538 [535]
121 [134]
315 [327]c;e 490 [494]d 571 [574]d
0.307 [0.273] )0.607 [)0.665] 0.960 [0.481]
201 [201]
2.189 [2.193]
[149]
[2.348]
a
Values for the Ac-PHGGGWGQ-NH2 are given in brackets. Parameters not obtained. c Nim ! Cu2þ charge transfer. d d–d transition. e N ! Cu2þ charge transfer. b
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 diminished negative ellipticity at 216 nm and positive signal at ca. 203 nm [30]. Paradoxically, a part of greater ellipticity at 226 nm, the pH 11 CD spectrum of the copper(II)–1 complex, resembles that of the free peptide ligand. 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, significant 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–d bands, 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 same pH 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 region that were interpreted in terms of simultaneous presence of copper (II) complexes having different magnetic parameters (Fig. 4). Unfortunately, the spectroscopic data do not allow the structural characterisation of the complex 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 cyclopeptide 2 could be responsible for such a different behaviour toward copper(II) binding. On the other hand, at pH 11 EPR signals attributable to a single major complex species are observed (Fig. 4). The measured magnetic 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, the pattern observed for the copper(II)–1 complex at the
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Table 3 Spectroscopic data for the copper(II) complexes of cyclo(GWGQPHGG) UV–Vis
pH
EPR
CD e (M1 cm1 )
k (nm)
k (nm)
De (M1 cm1 )
Ak
gk
5.0
780
17
6.0
707
29
7.0
656
68
349b 590c 693c
)0.027 )0.062 0.036
a
a
8.0
630
85
347b 590c 693c
)0.046 )0.100 0.095
a
a
9.0
570
91
324b; d 516c 595c 700c
0.100 )0.120 )0.061 0.100
a
a
10.0
546
115
326b; d 507c 592c
0.362 )0.334 0.143
a
a
11.0
552
132
318b; d 496c 584c
0.603 )0.705 0.891
199
2.190
a
Parameters not obtained. Nim ! Cu2þ charge transfer. c d–d transition. d N ! Cu2þ charge transfer. b
12
0.8
pH 5 pH 6
4
1.0
pH 10
0.6
∆ε (M-1 cm-1)
8
∆ε (M-1 cm-1)
1.2
pH 11
0 pH 9 pH 7
-4 pH 8
-8
pH 9
0.2
pH 5
0.0 -0.2
pH 6
-0.4
-0.8
190
200
210
220
230
240
250
260
300
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 region, (Fig. 7) it is evident that major effects on the cyclopeptideÕs 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
500
600
700
800
Wavelength
Wavelength Fig. 5. CD spectra of the copper(II)–1 complex in H2 O at different pH values (indicated on the curves).
400
pH 8
pH 10
pH 11
-0.6
-12 -16
pH 7
0.4
Fig. 6. Visible region CD spectra of the Copper(II)-2 complex with at different pH values (indicated on the curves).
responsible for the conformational changes is the complex species with deprotonated peptide nitrogen engaged in copper(II) binding. 4. Conclusion In the present study the copper(II) coordination properties of three prion peptide fragments have been comparatively investigated.
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193
References
∆ε (M-1 cm-1 )
10
0
-10 190
200
210
220
230
240
250
260
Wavelength (nm) Fig. 7. CD spectra of the copper(II)–2 complex in H2 O 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 behave similarly toward copper(II) complexation. From the spectroscopic data only two major complex species can be identified for the copper(II)–1 system: a 3N complex, 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-ordination sphere. Likewise, for the copper(II)–3 system the same set of donor atoms can be identified within the HGG 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 cyclopeptide 2 around pH 7 presumably through the histidineÕs imidazole side chain. However, the complex species formed around the neutrality and slightly basic pH values distinctly differ from those of the parent linear 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 RBNE01ARRA and MIUR PRIN 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 proteinÕs octarepeat model peptides Giuseppe Pappalardo a
a,*
, Giuseppe Impellizzeri b, Tiziana Campagna
a
Istituto CNR di Biostrutture e Bioimmagini-Sezione di Catania, V.le A. Doria 6, 95125 Catania, Italy b Dipartimento di Scienze Chimiche Universit a 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), AcPHGGGWGQ-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. Ó 2003 Elsevier B.V. All rights reserved. Keywords: Prion; Copper; Peptides; Metal complexes; Circular dichroism; EPR
1. Introduction The prion diseases, fatal neurodegenerative disorders, arise from the post-translational conversion of the normal cellular prion protein (PrPC ) into the pathological isoform PrPSc [1]. During this transformation PrPSc becomes infectious, protease-resistant, insoluble in non-denaturant detergents, and accumulates in affected brains [2]. PrPC and PrPSc differ only in their secondary and tertiary structures being increased b-sheet content and decreased a-helices in the pathological 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 normal 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: +39-095337678. E-mail address: [email protected] (G. Pappalardo). 0020-1693/$ - see front matter Ó 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 interaction. 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 interconverted in vitro by altering the metal ion occupancy [11]. The prion protein has also been proposed to function as a copper transport protein for internalisation 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],
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2.2. Peptide synthesis
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-terminus including the octarepeats is highly flexible and unstructured while the C-terminal region adopts a globular structure composed of three a-helices and two short bstrands [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) complex species with the Ac-PHGGGWGQ-NH2 and AcHGGG-NH2 peptides. The results led to the conclusion that, at neutral pH, metal coordination within an octarepeat arises from the His imidazole and amide nitrogens encompassing the HGGG peptide fragment [30]. More recently, the solid state structure of the Cu(II)– HGGGW complex has been reported. It shows, in addition to the expected involvement of the His imidazole and the peptide nitrogens, an indirect participation of the indole nitrogen from the Trp side chain to the coordination of the metal ion [31,32]. To obtain further information on the copper(II) complexÕs 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 chainÕs flexibility and primary structure are stringent parameters to provide the precise copper(II) coordination environment within a single octarepeat.
The synthesis and purification of Ac-PHGGGWGQNH2 (3) has been reported elsewhere [30]. The peptide Ac-GWGQPHGG-NH2 (1) was synthesised by solid phase 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 Cterminal carboxylate group was synthesised in the amide form, while the N-terminal amino group was acetylated with 0.3 M Ac2 O in DMF after completion of the synthesis. This was done to suppress participation 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 cyclisation 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 precursor and peptide 1 were cleaved off from the respective resins by treatment with a mixture of TFA/phenol/H2 O/ 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 semipreparative reversed-phase HPLC on a 250 10 mm pores). Each pepVydac C18 (5 lm particle size, 300-A 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 characterised by 1 H NMR spectroscopy and FAB-MS spectrometry [peptide 1: m=z 836.4 (M + H)þ calc. for C37 H49 N13 O10 835.37; peptide 2: 777.2 (M + H)þ calc. for C35 H44 N12 O9 776.33].
2. Experimental
2.3. Spectroscopic measurements
2.1. Materials
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 an aqueous solution of ammonium D -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 region (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 the average of 8–20 scans. CD intensities are expressed in De (M1 cm1 ).
All N-fluorenylmethyloxycarbonyl(Fmoc)-protected aminoacids, Novasyn-TGR resin, 2-(1-H-benzotriazole1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and N-hydroxybenzotriazole (HOBT), were purchased from Novabiochem (Switzerland). N,NDimethylformamide (DMF, peptide synthesis grade) and 20% piperidine/DMF solution, were from Applied Biosearch. N,N-Diisopropylethyl amine (DIEA), triisopropylsilane (TIS), ethanedithiol (EDT), thioanisole and trifluoroacetic acid (TFA) were from Sigma/Aldrich. All other chemicals were of the highest available grade and were used without further purification.
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2.3.3. ESR spectroscopy Frozen solution ESR spectra were recorded on a Bruker 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 63 Cu(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 to the 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 spectroscopies. 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 reported. Both peptides 1 and 3 display nearly the same CD 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 octarepeats 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 ellipticity 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-
10
∆ε (M-1 cm-1 )
2.3.2. NMR spectroscopy Five millimolar solution samples were prepared in 90/ 10 H2 O/D2 O or pure D2 O containing trimethylsilylpropionic acid (TSP) as the internal standard. The pH of the solution was adjusted to the desired value by adding the suitable acid or base solution. The measured electrode 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 and 512 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 experiments were acquired using a 80 ms mixing time.
187
2
0
3 1
-10
190
200
210
220 230 240 Wavelength (nm)
250
260
Fig. 1. CD spectra of Ac-GWGQPHGG-NH2 (1), C-(GWGQPHGG) (2) and Ac-PHGGGWGQ-NH2 (3) in H2 O, pH 7.
ally well-defined models of turn conformations of the peptide chain [35]. In the present case, the negative ellipticity at 232 nm and the positive band around 200 nm might be related with the presence of a b turn conformation [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 appreciably 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 experiments were recorded in aqueous solution at pH 4. Resonance assignments were achieved using g-COSY [37] and TOCSY [38] spectra and then sequential assignments were extracted by inspection of the dipolar connectivities in the NOESY [39] or ROESY [40] spectra. It should be said that in all the peptides here investigated 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 assignments reported in Table 1 are relevant to the predominant 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 results obtained 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
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Table 1 Resonance assignments for the linear (1 and 3) and cyclic (2) peptides Residue
NH
Peptide 1 Ac-GWGQPHGG-NH2 Ac Gly 8.14 Trp 8.01 Gly 8.33 Gln 8.01 Pro His 8.68 Gly 8.47 Gly 8.32 NH2 Peptide 2 cyclo-(GWGQPHGG) Gly 8.06 Trp 8.61 Gly 8.48 Gln 7.65 Pro His 8.50 Glya 8.28 Glya 8.02 Peptide 3 Ac-PHGGGWGQ-NH2 Ac Pro His 8.59 Gly 8.39 Glya 8.32 Glya 8.32 Trp 8.09 Gly 8.32 Gln 8.05 NH2 a
Ha
Hb
Others 1.93
3.82 4.62 3.80 4.58 4.38 4.65 3.97 3.92
3.31, 3.26
2H 7.25; 4H 7.62; 5H 7.17; 6H 7.25; 7H 7.50; NH 10.14
2.06, 1.89 2.26, 1.84 3.27, 3.18
cCH2 2.32; NH2 7.59, 6.87 cCH2 1.99; dCH2 3.76, 3.64 2H 8.57; 4H 7.31
7.53; 7.08 3.94 4.39 3.80, 3.42 4.65 4.36 4.68 4.21, 3.90 3.99
3.32
2H 7.28; 4H 7.64; 5H 7.20; 6H 7.27; 7H 7.54; NH 10.12
2.16, 1.84 2.22, 1.63 3.40, 3.26
cCH2 2.32; NH2 7.48, 6.85 2H 8.61; 4H 7.34
2.04 4.27 4.59 4.01 3.92 3.92 4.43 3.84, 3.76 4.27
2.16; 1.90 3.21, 3.12;
2H 8.49, 4H 7.19
3.31; 3.24
2H 7.23; 4H 7.61; 5H 7.16; 6H 7.23; 7H 7.48; NH 10.11
2.10; 1.91
cCH2 2.28; NH2 7.50, 6.83 7.61; 7.09
Interchangeable values.
chain flexibility in the prionÕs 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 certain degree of backbone flexibility also in the cyclic analogue 2; indeed, the measured 3 JNH–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 Gln4 dNN (i; i þ 1) and between the C–Ha of Trp2 and the N–H of Gly3 protons daN (i; i þ 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 geometry 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 CD curves 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 transitions (LMCT) that may occur from imidazole or deprotonated peptide nitrogens to the copper ion [46]. These induced dichroic bands are clearly visible starting from pH 6.0, thus suggesting the anchoring function of the histidine residue in the co-ordination of the metal ion. Interestingly, the Cu(II) complex of the single octarepeat peptide 3 has the same sign CD bands as the copper(II)–1 complex in the whole pH range investigated in this study (Fig. 3(b)). The only apparent differences are in the intensities of the two series of spectra with those of the copper(II)–3 complex being more intense 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,
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189
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 AcGWGQPHGG-NH2 ( ), c-(GWGQPHGG) ( ), and Ac-PHGGGWGQ-NH2 ( ) in 90% H2 O/10% D2 O. 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.
1.2 (a)
1.0 0.8 0.4
0.6
pH 7
pH 8 pH 9
0.2
0.2 0.0
-0.2
-0.2
pH 6 pH 10
-0.4 pH 11
-0.6 300
400
pH 9
pH 6
0.4
0.0
-0.8
(b)
pH 7; pH 8
1.0 0.8
0.6
∆ε (M-1 cm-1)
1.2
pH 10 pH 11
-0.4 -0.6
500
600
700
800
--0.8
300
400
500
600
700
800
Wavelength 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).
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stated that in the range of pH 6–8 the major species is the CuH-2 L complex which involves the imidazole nitrogen and two amide nitrogens from the two subsequent glycine residues. Such a complex species gave a CD spectrum with the characteristic CD bands due to electronic 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 the multiple octarepeat peptides in the same wavelength 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 the coordination geometry relative to single and multiple repeat peptides is negligible. Further support for this comes from EPR and UV–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 octarepeat 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 the formation 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 appeared in the literature [47], we hypothesise that in such complex species also the amide nitrogen of the histidine residue 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] allowed the conclusion that the differences seen in the CD intensities for the two copper(II)–1 and copper(II)–3 complexes may be correlated with the proximity of the Trp side chain to the copper(II) coordination site in the copper(II)–3 system.
(a)
1 3 2
2600
2800
3000
3200
3400
3600
G (b)
1 3 2
2600
2800
3000
3200
3400
3600
G 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.
CD spectra in the UV region have been recorded to establish 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 copper(II) on the main chain conformation of the octarepeat 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 solution conformation 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;
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191
Table 2 Spectroscopic data for the copper(II) complexes of Ac-GWGQPHGG-NH2 and Ac-PHGGGWGQ-NH2 a pH
UV–Vis
CD
k (nm)
e (M1 cm1 )
5.0
800 [790]
13 [16]
6.0
700 [645]
7.0
EPR
k (nm)
De (M1 cm1 )
Ak
gk
18 [26]
333 [338]c 588 [598]d 693 [727]d
0.042 [0.170] 0.036 [0.212] )0.021 [)0.094]
b
b
634 [625]
50 [87]
333 [337]c 584 [598]d 698 [717]d
0.257 [0.641] 0.184 [0.815] )0.162 [)0.331]
167 [165]
2.230 [2.236]
8.0
625 [622]
61 [87]
333 [337]c 584 [598]d 697 [717]d
0.395 [0.682] 0.268 [0.817] )0.294 [)0.336]
165 [164]
2.231 [2.237]
9.0
605 [605]
62 [79]
332 [335]c 500d 588 [598]d 697 [717]d
0.390 [0.570] )0.062 0.295 [0.560] )0.210 [)0.256]
166 [163]
2.232 [2.235]
10.0
550 [545]
92 [103]
322 [332]c;e 508 [498]d 593 [584]d 702d
0.280 [0.394] )0.254 [)0.181] 0.270 [0.159] )0.067
166 [b ]
2.230 [b ]
11.0
538 [535]
121 [134]
315 [327]c;e 490 [494]d 571 [574]d
0.307 [0.273] )0.607 [)0.665] 0.960 [0.481]
201 [201]
2.189 [2.193]
[149]
[2.348]
a
Values for the Ac-PHGGGWGQ-NH2 are given in brackets. Parameters not obtained. c Nim ! Cu2þ charge transfer. d d–d transition. e N ! Cu2þ charge transfer. b
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 diminished negative ellipticity at 216 nm and positive signal at ca. 203 nm [30]. Paradoxically, a part of greater ellipticity at 226 nm, the pH 11 CD spectrum of the copper(II)–1 complex, resembles that of the free peptide ligand. 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, significant 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–d bands, 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 same pH 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 region that were interpreted in terms of simultaneous presence of copper (II) complexes having different magnetic parameters (Fig. 4). Unfortunately, the spectroscopic data do not allow the structural characterisation of the complex 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 cyclopeptide 2 could be responsible for such a different behaviour toward copper(II) binding. On the other hand, at pH 11 EPR signals attributable to a single major complex species are observed (Fig. 4). The measured magnetic 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, the pattern observed for the copper(II)–1 complex at the
192
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Table 3 Spectroscopic data for the copper(II) complexes of cyclo(GWGQPHGG) UV–Vis
pH
EPR
CD e (M1 cm1 )
k (nm)
k (nm)
De (M1 cm1 )
Ak
gk
5.0
780
17
6.0
707
29
7.0
656
68
349b 590c 693c
)0.027 )0.062 0.036
a
a
8.0
630
85
347b 590c 693c
)0.046 )0.100 0.095
a
a
9.0
570
91
324b; d 516c 595c 700c
0.100 )0.120 )0.061 0.100
a
a
10.0
546
115
326b; d 507c 592c
0.362 )0.334 0.143
a
a
11.0
552
132
318b; d 496c 584c
0.603 )0.705 0.891
199
2.190
a
Parameters not obtained. Nim ! Cu2þ charge transfer. c d–d transition. d N ! Cu2þ charge transfer. b
12
0.8
pH 5 pH 6
4
1.0
pH 10
0.6
∆ε (M-1 cm-1)
8
∆ε (M-1 cm-1)
1.2
pH 11
0 pH 9 pH 7
-4 pH 8
-8
pH 9
0.2
pH 5
0.0 -0.2
pH 6
-0.4
-0.8
190
200
210
220
230
240
250
260
300
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 region, (Fig. 7) it is evident that major effects on the cyclopeptideÕs 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
500
600
700
800
Wavelength
Wavelength Fig. 5. CD spectra of the copper(II)–1 complex in H2 O at different pH values (indicated on the curves).
400
pH 8
pH 10
pH 11
-0.6
-12 -16
pH 7
0.4
Fig. 6. Visible region CD spectra of the Copper(II)-2 complex with at different pH values (indicated on the curves).
responsible for the conformational changes is the complex species with deprotonated peptide nitrogen engaged in copper(II) binding. 4. Conclusion In the present study the copper(II) coordination properties of three prion peptide fragments have been comparatively investigated.
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193
References
∆ε (M-1 cm-1 )
10
0
-10 190
200
210
220
230
240
250
260
Wavelength (nm) Fig. 7. CD spectra of the copper(II)–2 complex in H2 O 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 behave similarly toward copper(II) complexation. From the spectroscopic data only two major complex species can be identified for the copper(II)–1 system: a 3N complex, 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-ordination sphere. Likewise, for the copper(II)–3 system the same set of donor atoms can be identified within the HGG 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 cyclopeptide 2 around pH 7 presumably through the histidineÕs imidazole side chain. However, the complex species formed around the neutrality and slightly basic pH values distinctly differ from those of the parent linear 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 RBNE01ARRA and MIUR PRIN 2001 Project 2001031717_003 for financial support. Thanks are also due to Prof. Enrico Rizzarelli for helpful discussions.
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