This work provides evidence that paramagnetic lanthanide ions constitute ideal probes suitable for investigations of metal effects upon peptide–receptor interactions with the use of NMR methods. Cerium(III) is herein used for assessing metal effects upon the interaction between angiotensin II and a fragment from the AT1Areceptor. Angiotensin II forms a complex with cerium(III) in water while the fCT300–320receptor fragment is poorly affected by cerium(III). However, the addition of the fragment displaces cerium(III) from the complex, thus directly demonstrating the higher affinity of angiotensin II for the receptor and probing the peptide residues involved in receptor binding

Daniela Valensin

Elena Gaggelli

Elena Molteni

Francesca Mancini

Gianni Valensin

Nicola D'Amelio

Nicola Gaggelli

English

This work provides evidence that paramagnetic lanthanide ions constitute ideal probes suitable for investigations of metal effects upon peptide-receptor interactions with the use of NMR methods. Cerium(Ill) is herein used for assessing metal effects upon the interaction between angiotensin II and a fragment from the AT(1A) receptor. Angiotensin II forms a complex with cerium(Ill) in water while the fCT(300-320) receptor fragment is poorly affected by cerium(III). However, the addition of the fragment displaces cerium(III) from the complex, thus directly demonstrating the higher affinity of angiotensin II for the receptor and probing the peptide residues involved in receptor binding

N. D'AMELIO

GAGGELLI, ELENA

GAGGELLI, NICOLA

F. MANCINI

E. MOLTENI

VALENSIN, DANIELA

VALENSIN, GIANNI

Archivio della Ricerca - Università degli Studi di Siena

Probing the role of metal ions on reversible peptide-protein interactions by NMR

This work provides evidence that paramagnetic lanthanide ions constitute ideal probes suitable for investigations of metal effects upon peptide–receptor interactions with the use of NMR methods. Cerium(III) is herein used for assessing metal effects upon the interaction between angiotensin II and a fragment from the AT1Areceptor. Angiotensin II forms a complex with cerium(III) in water while the fCT300–320receptor fragment is poorly affected by cerium(III). However, the addition of the fragment displaces cerium(III) from the complex, thus directly demonstrating the higher affinity of angiotensin II for the receptor and probing the peptide residues involved in receptor binding.</jats:p

Crossref

Spectroscopy 18 (2004) 251–256 251IOS PressProbing the role of metal ions on reversiblepeptide–protein interactions by NMRNicola D’Amelio, Elena Gaggelli ∗, Nicola Gaggelli, Francesca Mancini, Elena Molteni,Daniela Valensin and Gianni ValensinDepartment of Chemistry and the NMR Center, University of Siena, Via A.Moro, Siena 53100, ItalyAbstract. This work provides evidence that paramagnetic lanthanide ions constitute ideal probes suitable for investigations ofmetal effects upon peptide–receptor interactions with the use of NMR methods. Cerium(III) is herein used for assessing metaleffects upon the interaction between angiotensin II and a fragment from the AT1A receptor. Angiotensin II forms a complexwith cerium(III) in water while the fCT300–320 receptor fragment is poorly affected by cerium(III). However, the addition ofthe fragment displaces cerium(III) from the complex, thus directly demonstrating the higher affinity of angiotensin II for thereceptor and probing the peptide residues involved in receptor binding.Keywords: NMR, 1H, angiotensin II, AT1A receptor, lanthanides, cerium(III)AbbreviationsCOSY, correlation spectroscopy; HPLC, high pressure liquid chromatography; MALDITOF, matrixassisted laser desorption/ionization time of flight; MRI, magnetic resonance imaging; NMR, nuclearmagnetic resonance; ROESY, rotating frame Overhauser effect spectroscopy; TOCSY, total correlationspectroscopy; TSP, 3-(trimethylsilyl)-[2,2,3,3-d4] propanosulphonate sodium salt; Ln, lanthanides.1. IntroductionPeptides are primary messengers for inter-cellular communication in biological systems. In contrastto DNA recognition, the information content is not encoded into the sequence, but depends on the three-dimensional structure. Since most peptides are intrinsically extremely flexible, such “active” structureresults from nearest-neighbor contacts with the protein receptor surface (conformational selection). Elec-trostatics is likely to play a major role in such a process and, as a consequence, charged metal ions areexpected to actively modulate the interaction.We herein provide evidence that paramagnetic lanthanide ions constitute ideal suitable probes for in-vestigations of metal effects upon peptide–receptor interactions with the use of NMR methods. As amatter of fact, lanthanides have already been successfully exploited for (i) probing the metal binding sitein metalloproteins [1], (ii) delineating exposed residues on protein surfaces [2,3], and (iii) enhancingcontrast in MRI [4–6]. We have considered the interaction of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, AngII), a peptide playing a critical role in regulation of cardiovascular homeostasis [7] with*Corresponding author. Tel.: +39 577 234243; Fax: +39 577 234254; E-mail: gaggelli@unisi.it.0712-4813/04/$17.00  2004 – IOS Press and the authors. All rights reserved252 N. D’Amelio et al. / Probing the role of metal ions on reversible peptide–protein interactions by NMRa sequence taken from the C-terminus (Leu-Phe-Tyr-Gly-Phe-Leu-Gly-Lys-Lys-Phe-Lys-Lys-Tyr-Phe-Leu-Gln-Leu-Leu-Lys-Tyr-Ile, fCT300–320) of its AT1 receptor [8], with the aim of ascertaining the po-tential of the method in cases of reversible associations. Ang II is long known to behave as a flexibleunstructured peptide in polar environments, but calcium [9] or lanthanide (III) ions [10] force the pep-tide to assume a folded hairpin configuration that discloses a striking similarity with one of the structuressuggested for bound Ang II [11]. It follows that the metal is likely to act as agonist or antagonist for theAng II–AT1 interaction.We used Ce(III) as the lanthanide because (i) its ionic radius is very close to that of Ca(II) and (ii) itslarge magnetic susceptibility causes large shifts of proton signals [1], thus amplifying the effects of metalbinding.2. ExperimentalAng II was purchased from Sigma Cheamical Co. as acetate and used without further purifica-tion. fCT300–320 was assembled using a Syro peptide synthesizer (MultiSynTech, Bochum, D) on aRink amide MBHA resin. Standard solid-phase chemistry based on the Fmoc protection group andDIC/HOBt activation was employed. The following side-chain protecting groups were used: t-butyl forTyr, t-butoxycarbonyl for Lys and trityl for Gln. The peptide was side-chain deprotected and cleavedfrom the resin by treatment with trifluoracetic acid (TFA) containing ethandithiol, water, triisobutyl-silane and anisole (93/2.5/2/1.5/1). After precipitation by ethylic ether, the crude peptide was purifiedby reverse-phase HPLC on a Vydac C18 column (1.0 × 25 cm), using a 30 min linear gradient from30 to 100% of methanol in water (both containing 0.1% of TFA). Purity and structural integrity of thepeptide were confirmed by analytical HPLC and by MALDITOF on a Biflex III spectrometer (Bruker,Breemen, D).NMR spectra were performed at 14.1 T with a Bruker Avance 600 MHz spectrometer at controlledtemperatures (±0.1 K). The peptides were dissolved in a solution of deionized water containing D2O(10%). The pH was adjusted at pH= 4.0 with either DCl or NaOD. The desired concentration of ceriumions was achieved by using a stock solution of cerium(III) chloride (Sigma Chemical Co.) in deionizedwater at pH 4.0. The experiments were carried out at the constant ionic strength I = 150 mM. The as-signment was obtained by TOCSY, COSY, and ROESY 2D experiments. TOCSY spectra were recordedwith a total spin-locking time of 75 ms using a MLEV-17 mixing sequence; ROESY spectra were per-formed with a standard pulse sequence using a ROESY spin-lock of 350 ms and a power of 2.3 kHz.During all 2D experiments, water suppression was achieved by excitation sculpting and presaturationmethods. The spectral width of homonuclear 2D experiments was typically 6600 Hz in both F1 and F2dimensions.3. Results and discussionAll NMR spectra were recorded at pH 4.0 and 318 K, conditions that provided the best lineshape andare reported to maximize the occurrence of secondary structure in fCT300–320 [12]. Chemical shifts aresummarized in Tables 1 and 2. Figure 1 shows the effects of adding Ce3+ on the NMR spectrum ofAng II: the most affected 1H signals belong to 8Phe. Among the others, the Hβ of 1Asp and the Hε of6His were also largely affected, as expected for both the Asp and terminal carboxyl groups binding themetal [9,10]. The most affected proton resonance (the amide proton of 8Phe) was used to evaluate theN. D’Amelio et al. / Probing the role of metal ions on reversible peptide–protein interactions by NMR 253Table 11H chemical shifts of Ang II 2.8 mM in H2O (containing 10% D2O) at pH 4.0, T = 318 K. Chemical shifts were referenced tointernal TSP1Asp Hα 4.30Hβ 2.87, 2.742Arg HN –Hα 4.36Hβ 1.75Hγ 1.54, 1.53Hδ 3.17HNsc 7.06, 6.593Val HN 7.99Hα 4.09Hβ 1.99Hγ 0.87, 0.834Tyr HN 8.26Hα 4.63Hβ 2.98, 2.88Hδ 7.09Hε 6.775Ile HN 7.86Hα 4.10Hβ 1.73Hγ 1.33, 1.11Hδ 0.796His HN 8.22Hα 4.80Hβ 3.17, 3.09Hδ 7.22Hε 8.517Pro Hα 4.40Hβ 2.22, 1.95Hγ 1.95Hδ 3.74, 3.558Phe HN 7.53Hα 4.50Hβ 3.18, 3.06Hδ 7.26Hε 7.33Hz 7.26Table 21H chemical shifts of backbone and aromatic protons of fCT300–320 2 mM in H2O (containing 10% D2O) at pH 4.0, T = 318 K.Chemical shifts were referenced to internal TSP1Leu HN –Hα 3.872Phe HNHα 4.49Hδ 7.19Hε 7.29Hz –3Tyr HN 8.03Hα 4.43Hδ 7.01Hε 6.814Gly HN 7.50Hα 3.745Phe HN 7.79Hα 4.53Hδ 7.20Hε 7.28Hz 7.306Leu HN 7.88Hα 4.257Gly HN 7.91Hα 3.998Lys HN 8.15Hα 4.169Lys HN 8.15Hα 4.1710Phe HN 7.97Hα 4.50Hδ 7.19Hε 7.23Hz –11Lys HN 8.01Hα 4.0112Lys HN 7.94Hα 4.0913Tyr HN 7.78Hα 4.43Hδ 6.99Hε 6.7814Phe HN 8.28Hα 4.29Hδ 7.15Hε 6.85Hz 7.2415Leu HN 8.09Hα 4.0716Gln HN 7.85Hα 4.1317Leu HN 7.89Hα 4.1218Leu HN 7.86Hα 4.0419Lys HN 7.70Hα 4.0820Tyr HN 7.81Hα 4.50Hδ 7.07Hε 6.8421Ile HN 7.74Hα 4.05254 N. D’Amelio et al. / Probing the role of metal ions on reversible peptide–protein interactions by NMRFig. 1. Low- (a) and high-field (b) regions of superimposed 1H-NMR spectra of angiotensin II 5.00 mM in H2O/D2O 9/1 in theabsence (bottom) and in the presence (top) of Ce3+ 0.125 M at T = 318 K, pH = 4.0. 8Phe HN (empty square), Hα (emptyrhombus), Hβ (empty circle), Hδ (cross) and Hε (filled square), 1Asp Hβ (filled circle) and 6His Hε (filled rhombus) displayshift.dissociation constant (Kd = 33.8±2 mM) and the stoichiometry of the metal complex (n = 1.01±0.03)by using reported methods [13,14].In contrast with Ang II, the fCT fragment was poorly affected by the addition of Ce3+. Small(ca. 0.05 ppm) downfield shifts were observed for almost all amide protons, thus suggesting that this21-mer does not provide any specific binding site for the lanthanide ion. These findings were not unex-pected due to lacking of Asp or Glu residues that usually act as anchoring sites for calcium and Ln(III).Upon binding to the fCT fragment, all amide protons of Ang II, especially the 8Phe HN, were shifteddownfield (0.05–0.15 ppm at fCT : Ang II 1 : 2) and, at a lesser extent, the same occurred for 6His and8Phe aromatic protons [15]. The shifts experienced by HN, Hα and aromatic protons of fCT allowed tolocate the interaction site at level of the N-terminus, involving the 1Asp and 8Phe of Ang II and implyinga well defined pairing pattern of aromatic rings [15]. For the present purposes, it is worth underliningthat the shifts induced by Ce3+ were two-, three-fold larger than those determined by the addition of fCT.It was then observed that addition of fCT 1.0 mM to Ang II 0.25 mM almost completely quenchedthe paramagnetic shifts induced by Ce3+ 0.50 mM (Fig. 2). Such behavior may be in principle deter-mined by:N. D’Amelio et al. / Probing the role of metal ions on reversible peptide–protein interactions by NMR 255Fig. 2. Superimposed 1H-NMR spectra of angiotensin II 0.25 mM 5 in H2O/D2O 9/1 alone (bottom), in the presence of Ce3+0.50 mM (center), in the presence of Ce3+ 0.50 mM and of fCT300–320 1.00 mM (top) at T = 318 K, pH = 4.0. The arrowsshow the shift induced by Ce3+ on 8Phe HN and the quenching effect caused by the addition of fCT300–320.(i) formation of a new (ternary) complex, in fast exchange with the old one, with a reoriented magneticsusceptibility tensor of the lanthanide that changes the sign of observed shifts;(ii) competition of fCT and Ang II for Ce3+ that is displaced from its original binding site;(iii) competition of fCT and Ce3+ for Ang II: the lanthanide is displaced from its original bindingsites on Ang II because the same groups are now engaged in a (more) specific interaction with thereceptor peptide.At the light of the observed poor affinity of Ce3+ for the fCT fragment, we believe hypothesis (iii)is the most reliable and the simplest in accounting for the observed phenomena. That this is the case isfurther supported by the observation that 1Asp and 8Phe of Ang II are both involved in binding eitherCe3+ or fCT.It is therefore concluded that paramagnetic lanthanide ions provide a simple NMR tool that may addsome valuable information on the role metal ions, especially calcium, play in reversible peptide–receptorinteractions. The value of the dissociation constant and the consequent fast exchange regime (the chem-ical shift induced by the metal is a linear function of the molar fraction of the bound form) assure thatthe complex is kinetically labile and thus well suited to investigate whether and how its formation andstructure are affected by the presence of the receptor for the peptide. The observed canceling of para-magnetic effects upon addition of the fCT300–320 fragment is therefore directly probing not only thatAng II obviously has a larger affinity for the receptor than for the metal but also that the metal and thereceptor compete for the same donors in the peptide.The same approach is expected to encompass all cases where the paramagnetic ion finds a muchmore specific binding site in one than in the other of the interacting pair; whereas the reversibility ofthe peptide–receptor interaction is not probably of concern. In the considered example, since the metal256 N. D’Amelio et al. / Probing the role of metal ions on reversible peptide–protein interactions by NMRforces Ang II to assume a conformation resembling the hairpin suggested as a possible biological activestructure [11], it may be speculated that the metal ion aids in selecting an active structure of the peptidewithout severely affecting the interaction modes at the biological target.AcknowledgementWe would like to acknowledge the CIRMMP (Consorzio Interuniversitario Risonanze Magnetiche diMetalloproteine Paramagnetiche) for financial support.References[1] I. Bertini, C. Luchinat and G. Parigi, Magnetic susceptibility in paramagnetic NMR, Progr. NMR Spectrosc. 40 (2002),249–273, and references therein.[2] L.R. Dick, C.F.G.C. Geraldes, A.D. Sherry, C.W. Gray and D.M. Gray, 13C NMR of methylated lysines of fd gene5 protein: evidence for a conformational change involving lysine 24 upon binding of a negatively charged lanthanidechelate, Biochemistry 28 (1989), 7896–7904.[3] S. Aime, N. D’Amelio, M. Fragai, Y.M. Lee, C. Luchinat, E. Terreno and G. Valensin, A paramagnetic probe to localizeresidues next to carboxylates on protein surfaces, J. Biol. Inorg. Chem. 7 (2002), 617–622.[4] J.A. Peters, J. Huskens and D.J. Raber, Lanthanide induced shifts and relaxation rate enhancements, Progr. NMR Spec-trosc. 28 (1996), 283–350.[5] S. Aime, M. Botta, M. Fasano and E. Terreno, Lanthanide(III) chelates for NMR biomedical applications, Chem. Soc. Rev.27 (1998), 19–29.[6] P. Caravan, J.J. Ellison, T.J. McMurry and R.B. Lauffer, Gadolinium(III) chelates as MRI contrast agents: structure,dynamics, and applications, Chem. Rev. 99 (1999), 2293–2352.[7] M. De Gasparo, K.J. Catt, T. Inagami, J.W. Wright and T.H. Unger, International union of pharmacology. XXIII. Theangiotensin II receptors, Pharmacol. Rev. 52 (2000), 415–472.[8] P.B. Timmermans, P.C. Wong, A.T. Chiu and W.F. Herblin, Nonpeptide angiotensin II receptor antagonists, Trends Phar-macol. Sci. 12 (1991), 55–62.[9] E. Gaggelli, N. D’Amelio, N. Gaggelli, D. Valensin, A. Maccotta and G. Valensin, NMR studies of calcium complexes ofbiological active peptides, Recent Res. Develop. Inorganic. Chem. 2 (2000), 131–144.[10] R.E. Lenkinski and R.L. Stephens, The nature of the Ln3+–angiotensin II complex. A 13C nmr study of the binding ofYb3+ to angiotensin II, J. Inorg. Biochem. 18 (1983), 175–180.[11] K.C. Garcia, P.M. Ronco, P.J. Verroust, A.T. Brunger and L.M. Amzel, Three-dimensional structure of an angiotensinII-Fab complex at 3 A: hormone recognition by an anti-idiotypic antibody, Science 257 (1992), 502–507.[12] L. Franzoni, G. Nicastro, T.A. Perthinez, M. Tatò, C.R. Nakaie, A.C.M. Paiva, S. Schreier and A. Spisni, Structure of theC-terminal fragment 300–320 of the rat angiotensin II AT1A receptor and its relevance with respect to G-protein coupling,J. Biol. Chem. 272 (1997), 9734–9741.[13] J. Reuben, Complex formation betwee Eu(fod)3, a lanthanide shift reagent, and organic molecules, J. Am. Chem. Soc. 95(1973), 3534–3540.[14] C.K. Vishwanath and K.R. Easwaran, Hydrogen-1 and carbon-13 magnetic resonance studies of nonactin–calcium com-plex, Biochemistry 20 (1981), 2018–2023.[15] N. D’Amelio, E. Gaggelli, N. Gaggelli, L. Lozzi, P. Neri, D. Valensin and G. Valensin, Interaction of angiotensin II withthe C-terminal 300–320 fragment of the rat angiotensin II receptor AT1a monitored by NMR, Biopolymers (2003), inpress.Submit your manuscripts athttp://www.hindawi.comHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Inorganic ChemistryInternational Journal ofHindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal ofPhotoenergyHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Carbohydrate ChemistryInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Journal ofChemistryHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Advances inPhysical ChemistryHindawi Publishing Corporationhttp://www.hindawi.com Analytical Methods in ChemistryJournal ofVolume 2014Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014SpectroscopyInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014Medicinal ChemistryInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014 Chromatography  Research InternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Applied ChemistryJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Theoretical ChemistryJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Journal ofSpectroscopyAnalytical ChemistryInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Quantum ChemistryHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014 Organic Chemistry InternationalElectrochemistryInternational Journal ofHindawi Publishing Corporation http://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014CatalystsJournal of

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Probing the role of metal ions on reversible peptide–protein interactions by NMR

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Spectroscopy 18 (2004) 251–256 251IOS PressProbing the role of metal ions on reversiblepeptide–protein interactions by NMRNicola D’Amelio, Elena Gaggelli ∗, Nicola Gaggelli, Francesca Mancini, Elena Molteni,Daniela Valensin and Gianni ValensinDepartment of Chemistry and the NMR Center, University of Siena, Via A.Moro, Siena 53100, ItalyAbstract. This work provides evidence that paramagnetic lanthanide ions constitute ideal probes suitable for investigations ofmetal effects upon peptide–receptor interactions with the use of NMR methods. Cerium(III) is herein used for assessing metaleffects upon the interaction between angiotensin II and a fragment from the AT1A receptor. Angiotensin II forms a complexwith cerium(III) in water while the fCT300–320 receptor fragment is poorly affected by cerium(III). However, the addition ofthe fragment displaces cerium(III) from the complex, thus directly demonstrating the higher affinity of angiotensin II for thereceptor and probing the peptide residues involved in receptor binding.Keywords: NMR, 1H, angiotensin II, AT1A receptor, lanthanides, cerium(III)AbbreviationsCOSY, correlation spectroscopy; HPLC, high pressure liquid chromatography; MALDITOF, matrixassisted laser desorption/ionization time of flight; MRI, magnetic resonance imaging; NMR, nuclearmagnetic resonance; ROESY, rotating frame Overhauser effect spectroscopy; TOCSY, total correlationspectroscopy; TSP, 3-(trimethylsilyl)-[2,2,3,3-d4] propanosulphonate sodium salt; Ln, lanthanides.1. IntroductionPeptides are primary messengers for inter-cellular communication in biological systems. In contrastto DNA recognition, the information content is not encoded into the sequence, but depends on the three-dimensional structure. Since most peptides are intrinsically extremely flexible, such “active” structureresults from nearest-neighbor contacts with the protein receptor surface (conformational selection). Elec-trostatics is likely to play a major role in such a process and, as a consequence, charged metal ions areexpected to actively modulate the interaction.We herein provide evidence that paramagnetic lanthanide ions constitute ideal suitable probes for in-vestigations of metal effects upon peptide–receptor interactions with the use of NMR methods. As amatter of fact, lanthanides have already been successfully exploited for (i) probing the metal binding sitein metalloproteins [1], (ii) delineating exposed residues on protein surfaces [2,3], and (iii) enhancingcontrast in MRI [4–6]. We have considered the interaction of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, AngII), a peptide playing a critical role in regulation of cardiovascular homeostasis [7] with*Corresponding author. Tel.: +39 577 234243; Fax: +39 577 234254; E-mail: gaggelli@unisi.it.0712-4813/04/$17.00  2004 – IOS Press and the authors. All rights reserved252 N. D’Amelio et al. / Probing the role of metal ions on reversible peptide–protein interactions by NMRa sequence taken from the C-terminus (Leu-Phe-Tyr-Gly-Phe-Leu-Gly-Lys-Lys-Phe-Lys-Lys-Tyr-Phe-Leu-Gln-Leu-Leu-Lys-Tyr-Ile, fCT300–320) of its AT1 receptor [8], with the aim of ascertaining the po-tential of the method in cases of reversible associations. Ang II is long known to behave as a flexibleunstructured peptide in polar environments, but calcium [9] or lanthanide (III) ions [10] force the pep-tide to assume a folded hairpin configuration that discloses a striking similarity with one of the structuressuggested for bound Ang II [11]. It follows that the metal is likely to act as agonist or antagonist for theAng II–AT1 interaction.We used Ce(III) as the lanthanide because (i) its ionic radius is very close to that of Ca(II) and (ii) itslarge magnetic susceptibility causes large shifts of proton signals [1], thus amplifying the effects of metalbinding.2. ExperimentalAng II was purchased from Sigma Cheamical Co. as acetate and used without further purifica-tion. fCT300–320 was assembled using a Syro peptide synthesizer (MultiSynTech, Bochum, D) on aRink amide MBHA resin. Standard solid-phase chemistry based on the Fmoc protection group andDIC/HOBt activation was employed. The following side-chain protecting groups were used: t-butyl forTyr, t-butoxycarbonyl for Lys and trityl for Gln. The peptide was side-chain deprotected and cleavedfrom the resin by treatment with trifluoracetic acid (TFA) containing ethandithiol, water, triisobutyl-silane and anisole (93/2.5/2/1.5/1). After precipitation by ethylic ether, the crude peptide was purifiedby reverse-phase HPLC on a Vydac C18 column (1.0 × 25 cm), using a 30 min linear gradient from30 to 100% of methanol in water (both containing 0.1% of TFA). Purity and structural integrity of thepeptide were confirmed by analytical HPLC and by MALDITOF on a Biflex III spectrometer (Bruker,Breemen, D).NMR spectra were performed at 14.1 T with a Bruker Avance 600 MHz spectrometer at controlledtemperatures (±0.1 K). The peptides were dissolved in a solution of deionized water containing D2O(10%). The pH was adjusted at pH = 4.0 with either DCl or NaOD. The desired concentration of ceriumions was achieved by using a stock solution of cerium(III) chloride (Sigma Chemical Co.) in deionizedwater at pH 4.0. The experiments were carried out at the constant ionic strength I = 150 mM. The as-signment was obtained by TOCSY, COSY, and ROESY 2D experiments. TOCSY spectra were recordedwith a total spin-locking time of 75 ms using a MLEV-17 mixing sequence; ROESY spectra were per-formed with a standard pulse sequence using a ROESY spin-lock of 350 ms and a power of 2.3 kHz.During all 2D experiments, water suppression was achieved by excitation sculpting and presaturationmethods. The spectral width of homonuclear 2D experiments was typically 6600 Hz in both F1 and F2dimensions.3. Results and discussionAll NMR spectra were recorded at pH 4.0 and 318 K, conditions that provided the best lineshape andare reported to maximize the occurrence of secondary structure in fCT300–320 [12]. Chemical shifts aresummarized in Tables 1 and 2. Figure 1 shows the effects of adding Ce3+ on the NMR spectrum ofAng II: the most affected 1H signals belong to 8Phe. Among the others, the Hβ of 1Asp and the Hε of6His were also largely affected, as expected for both the Asp and terminal carboxyl groups binding themetal [9,10]. The most affected proton resonance (the amide proton of 8Phe) was used to evaluate theN. D’Amelio et al. / Probing the role of metal ions on reversible peptide–protein interactions by NMR 253Table 11H chemical shifts of Ang II 2.8 mM in H2O (containing 10% D2O) at pH 4.0, T = 318 K. Chemical shifts were referenced tointernal TSP1Asp Hα 4.30Hβ 2.87, 2.742Arg HN –Hα 4.36Hβ 1.75Hγ 1.54, 1.53Hδ 3.17HNsc 7.06, 6.593Val HN 7.99Hα 4.09Hβ 1.99Hγ 0.87, 0.834Tyr HN 8.26Hα 4.63Hβ 2.98, 2.88Hδ 7.09Hε 6.775Ile HN 7.86Hα 4.10Hβ 1.73Hγ 1.33, 1.11Hδ 0.796His HN 8.22Hα 4.80Hβ 3.17, 3.09Hδ 7.22Hε 8.517Pro Hα 4.40Hβ 2.22, 1.95Hγ 1.95Hδ 3.74, 3.558Phe HN 7.53Hα 4.50Hβ 3.18, 3.06Hδ 7.26Hε 7.33Hz 7.26Table 21H chemical shifts of backbone and aromatic protons of fCT300–320 2 mM in H2O (containing 10% D2O) at pH 4.0, T = 318 K.Chemical shifts were referenced to internal TSP1Leu HN –Hα 3.872Phe HNHα 4.49Hδ 7.19Hε 7.29Hz –3Tyr HN 8.03Hα 4.43Hδ 7.01Hε 6.814Gly HN 7.50Hα 3.745Phe HN 7.79Hα 4.53Hδ 7.20Hε 7.28Hz 7.306Leu HN 7.88Hα 4.257Gly HN 7.91Hα 3.998Lys HN 8.15Hα 4.169Lys HN 8.15Hα 4.1710Phe HN 7.97Hα 4.50Hδ 7.19Hε 7.23Hz –11Lys HN 8.01Hα 4.0112Lys HN 7.94Hα 4.0913Tyr HN 7.78Hα 4.43Hδ 6.99Hε 6.7814Phe HN 8.28Hα 4.29Hδ 7.15Hε 6.85Hz 7.2415Leu HN 8.09Hα 4.0716Gln HN 7.85Hα 4.1317Leu HN 7.89Hα 4.1218Leu HN 7.86Hα 4.0419Lys HN 7.70Hα 4.0820Tyr HN 7.81Hα 4.50Hδ 7.07Hε 6.8421Ile HN 7.74Hα 4.05254 N. D’Amelio et al. / Probing the role of metal ions on reversible peptide–protein interactions by NMRFig. 1. Low- (a) and high-field (b) regions of superimposed 1H-NMR spectra of angiotensin II 5.00 mM in H2O/D2O 9/1 in theabsence (bottom) and in the presence (top) of Ce3+ 0.125 M at T = 318 K, pH = 4.0. 8Phe HN (empty square), Hα (emptyrhombus), Hβ (empty circle), Hδ (cross) and Hε (filled square),1Asp Hβ (filled circle) and6His Hε (filled rhombus) displayshift.dissociation constant (Kd = 33.8±2 mM) and the stoichiometry of the metal complex (n = 1.01±0.03)by using reported methods [13,14].In contrast with Ang II, the fCT fragment was poorly affected by the addition of Ce3+. Small(ca. 0.05 ppm) downfield shifts were observed for almost all amide protons, thus suggesting that this21-mer does not provide any specific binding site for the lanthanide ion. These findings were not unex-pected due to lacking of Asp or Glu residues that usually act as anchoring sites for calcium and Ln(III).Upon binding to the fCT fragment, all amide protons of Ang II, especially the 8Phe HN, were shifteddownfield (0.05–0.15 ppm at fCT : Ang II 1 : 2) and, at a lesser extent, the same occurred for 6His and8Phe aromatic protons [15]. The shifts experienced by HN, Hα and aromatic protons of fCT allowed tolocate the interaction site at level of the N-terminus, involving the 1Asp and 8Phe of Ang II and implyinga well defined pairing pattern of aromatic rings [15]. For the present purposes, it is worth underliningthat the shifts induced by Ce3+ were two-, three-fold larger than those determined by the addition of fCT.It was then observed that addition of fCT 1.0 mM to Ang II 0.25 mM almost completely quenchedthe paramagnetic shifts induced by Ce3+ 0.50 mM (Fig. 2). Such behavior may be in principle deter-mined by:N. D’Amelio et al. / Probing the role of metal ions on reversible peptide–protein interactions by NMR 255Fig. 2. Superimposed 1H-NMR spectra of angiotensin II 0.25 mM 5 in H2O/D2O 9/1 alone (bottom), in the presence of Ce3+0.50 mM (center), in the presence of Ce3+ 0.50 mM and of fCT300–320 1.00 mM (top) at T = 318 K, pH = 4.0. The arrowsshow the shift induced by Ce3+ on 8Phe HN and the quenching effect caused by the addition of fCT300–320.(i) formation of a new (ternary) complex, in fast exchange with the old one, with a reoriented magneticsusceptibility tensor of the lanthanide that changes the sign of observed shifts;(ii) competition of fCT and Ang II for Ce3+ that is displaced from its original binding site;(iii) competition of fCT and Ce3+ for Ang II: the lanthanide is displaced from its original bindingsites on Ang II because the same groups are now engaged in a (more) specific interaction with thereceptor peptide.At the light of the observed poor affinity of Ce3+ for the fCT fragment, we believe hypothesis (iii)is the most reliable and the simplest in accounting for the observed phenomena. That this is the case isfurther supported by the observation that 1Asp and 8Phe of Ang II are both involved in binding eitherCe3+ or fCT.It is therefore concluded that paramagnetic lanthanide ions provide a simple NMR tool that may addsome valuable information on the role metal ions, especially calcium, play in reversible peptide–receptorinteractions. The value of the dissociation constant and the consequent fast exchange regime (the chem-ical shift induced by the metal is a linear function of the molar fraction of the bound form) assure thatthe complex is kinetically labile and thus well suited to investigate whether and how its formation andstructure are affected by the presence of the receptor for the peptide. The observed canceling of para-magnetic effects upon addition of the fCT300–320 fragment is therefore directly probing not only thatAng II obviously has a larger affinity for the receptor than for the metal but also that the metal and thereceptor compete for the same donors in the peptide.The same approach is expected to encompass all cases where the paramagnetic ion finds a muchmore specific binding site in one than in the other of the interacting pair; whereas the reversibility ofthe peptide–receptor interaction is not probably of concern. In the considered example, since the metal256 N. D’Amelio et al. / Probing the role of metal ions on reversible peptide–protein interactions by NMRforces Ang II to assume a conformation resembling the hairpin suggested as a possible biological activestructure [11], it may be speculated that the metal ion aids in selecting an active structure of the peptidewithout severely affecting the interaction modes at the biological target.AcknowledgementWe would like to acknowledge the CIRMMP (Consorzio Interuniversitario Risonanze Magnetiche diMetalloproteine Paramagnetiche) for financial support.References[1] I. Bertini, C. Luchinat and G. Parigi, Magnetic susceptibility in paramagnetic NMR, Progr. NMR Spectrosc. 40 (2002),249–273, and references therein.[2] L.R. Dick, C.F.G.C. Geraldes, A.D. Sherry, C.W. Gray and D.M. Gray, 13C NMR of methylated lysines of fd gene5 protein: evidence for a conformational change involving lysine 24 upon binding of a negatively charged lanthanidechelate, Biochemistry 28 (1989), 7896–7904.[3] S. Aime, N. D’Amelio, M. Fragai, Y.M. Lee, C. Luchinat, E. Terreno and G. Valensin, A paramagnetic probe to localizeresidues next to carboxylates on protein surfaces, J. Biol. Inorg. Chem. 7 (2002), 617–622.[4] J.A. Peters, J. Huskens and D.J. Raber, Lanthanide induced shifts and relaxation rate enhancements, Progr. NMR Spec-trosc. 28 (1996), 283–350.[5] S. Aime, M. Botta, M. Fasano and E. Terreno, Lanthanide(III) chelates for NMR biomedical applications, Chem. Soc. Rev.27 (1998), 19–29.[6] P. Caravan, J.J. Ellison, T.J. McMurry and R.B. Lauffer, Gadolinium(III) chelates as MRI contrast agents: structure,dynamics, and applications, Chem. Rev. 99 (1999), 2293–2352.[7] M. De Gasparo, K.J. Catt, T. Inagami, J.W. Wright and T.H. Unger, International union of pharmacology. XXIII. Theangiotensin II receptors, Pharmacol. Rev. 52 (2000), 415–472.[8] P.B. Timmermans, P.C. Wong, A.T. Chiu and W.F. Herblin, Nonpeptide angiotensin II receptor antagonists, Trends Phar-macol. Sci. 12 (1991), 55–62.[9] E. Gaggelli, N. D’Amelio, N. Gaggelli, D. Valensin, A. Maccotta and G. Valensin, NMR studies of calcium complexes ofbiological active peptides, Recent Res. Develop. Inorganic. Chem. 2 (2000), 131–144.[10] R.E. Lenkinski and R.L. Stephens, The nature of the Ln3+–angiotensin II complex. A 13C nmr study of the binding ofYb3+ to angiotensin II, J. Inorg. Biochem. 18 (1983), 175–180.[11] K.C. Garcia, P.M. Ronco, P.J. Verroust, A.T. Brunger and L.M. Amzel, Three-dimensional structure of an angiotensinII-Fab complex at 3 A: hormone recognition by an anti-idiotypic antibody, Science 257 (1992), 502–507.[12] L. Franzoni, G. Nicastro, T.A. Perthinez, M. Tatò, C.R. Nakaie, A.C.M. Paiva, S. Schreier and A. Spisni, Structure of theC-terminal fragment 300–320 of the rat angiotensin II AT1A receptor and its relevance with respect to G-protein coupling,J. Biol. Chem. 272 (1997), 9734–9741.[13] J. Reuben, Complex formation betwee Eu(fod)3, a lanthanide shift reagent, and organic molecules, J. Am. Chem. Soc. 95(1973), 3534–3540.[14] C.K. Vishwanath and K.R. Easwaran, Hydrogen-1 and carbon-13 magnetic resonance studies of nonactin–calcium com-plex, Biochemistry 20 (1981), 2018–2023.[15] N. D’Amelio, E. Gaggelli, N. Gaggelli, L. Lozzi, P. Neri, D. Valensin and G. Valensin, Interaction of angiotensin II withthe C-terminal 300–320 fragment of the rat angiotensin II receptor AT1a monitored by NMR, Biopolymers (2003), inpress.Submit your manuscripts athttp://www.hindawi.comHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Inorganic ChemistryInternational Journal ofHindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal ofPhotoenergyHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Carbohydrate ChemistryInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Journal ofChemistryHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Advances inPhysical ChemistryHindawi Publishing Corporationhttp://www.hindawi.com Analytical Methods in ChemistryJournal ofVolume 2014Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014SpectroscopyInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014Medicinal ChemistryInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014 Chromatography  Research InternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Applied ChemistryJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Theoretical ChemistryJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Journal ofSpectroscopyAnalytical ChemistryInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Quantum ChemistryHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014 Organic Chemistry InternationalElectrochemistryInternational Journal ofHindawi Publishing Corporation http://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014CatalystsJournal of

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Probing the role of metal ions on reversible peptide–protein interactions by NMR

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