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Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl Matthieu Starck,†,‡ Nathalie Sisommay,†,‡ Fanny A. Laporte,†,‡ Stéphane Oros,†,‡ Colette Lebrun,†,‡ and Pascale Delangle*,†,‡ †

Université Grenoble Alpes, INAC-SCIB, F-38000 Grenoble, France CEA, INAC-SCIB, F-38000 Grenoble, France

‡

S Supporting Information *

ABSTRACT: Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a β-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 1010 at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity.

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was demonstrated by Vidaud et al.24to bind uranyl tightly, with nine uranyl ions bound in human OPN. Interestingly, surface plasmon resonance studies demonstrated that phosphorylation is crucial for a high uranyl affinity: a loss of 1−2 orders of magnitude is evidenced upon dephosphorylation of the proteins. The high-affinity uranyl-binding sites of OPN have not been identified; however, the polyphosphorylated sequence pSDEpSDE is thought to be involved in bone mineralization26 and in calcium and uranyl binding due to both phosphate and carboxylate groups.27 We take advantage of preorganized peptide scaffolds recently validated in our group as efficient uranyl-binding peptides to help in elucidating uranyl coordination properties of phosphorylated proteins such as OPN, which is proposed to be a privileged target of this ion in vivo. Cyclodecapeptides that contain two prolylglycine (Pro-Gly) sequences as β-turn inducers form an antiparallel β-sheet structure28,29 that has been exploited for the design of copper-chelating agents30−32 and more recently uranyl-binding peptides.33 The nuclear magnetic resonance solution structure of such peptides in water shows the preorientation of the four side chains at positions 1, 3, 6, and 8 to coordinate metal ions in the upper face of the peptide scaffold.29 For instance, peptide A1 fits perfectly to uranyl coordination because of four acidic side chains of glutamic acids that converge in the equatorial plane of the dioxo cation, to give the well-defined UO2·A1 complex with the β-sheet structure represented in Scheme 1.33 Importantly, the

INTRODUCTION Uranium belongs to the actinide series and is a natural element widely found in the environment, because of its natural occurrence and industrial applications, especially in energy production. Although the existence of uranium toxicity has been known for a long time, there is still a serious lack of knowledge about the underlying molecular interactions in vivo.1−3 Whatever the route of entry, uranium reaches the blood and then targets mainly the kidneys and the bones.4,5 Current therapies for the treatment of uranium contamination are limited, and extensive research has to be dedicated to the identification of uranyl targets in vivo and the development of efficient chelators for medical use.5−7 The relevant form under physiological conditions is the hexavalent uranyl cation, UO22+, which is classified as a hard Lewis acid and is highly oxophilic with a high affinity for hard oxygen donors. The presence of the two oxo groups in this rare form of linear trans dioxo cation favors the coordination of four to six extra ligands in the equatorial plane, which is perpendicular to the O−U−O bonds. Therefore, the preferred binding sites of uranyl in proteins involve hard oxygen donors from carboxylates, phenolates, and phosphates.8 In particular, phosphate functions in peptides or proteins are known to be very efficient binding groups for hard cations such as f-elements.9−11 Several uranyl target proteins have been identified over the past few decades.12−18 Among them are the two major serum proteins, albumin and transferrin,3,19−22 but also less abundant proteins involved in bone mineralization, which display a particularly high affinity for uranyl.23,24 The highly phosphorylated protein osteopontin (OPN),25 which plays an important role in bone homeostasis, © 2015 American Chemical Society

Received: September 30, 2015 Published: November 19, 2015 11557

DOI: 10.1021/acs.inorgchem.5b02249 Inorg. Chem. 2015, 54, 11557−11562

Article

Inorganic Chemistry Scheme 1. Prototype Starting Complex UO2·A1 and Cyclodecapeptides Mimicking OPN-Binding Sites Studied Herein

chromatography (HPLC) are solvent A [99.925/0.075 (v/v) H2O/ trifluoroacetic acid (TFA)] and solvent B [90/10/0.1 (v/v/v) CH3CN/H2O/TFA]. Linear Precursor 1. H-Ser(PO3HBz)-Arg(Pbf)-Glu(tBu)-Pro-GlyGlu(tBu)-Trp(Boc)-Ser(PO3HBz)-Pro-Gly-OH (Bz = benzyl, Pbf = 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, tBu = tert-butyl, and Boc = tert-butoxycarboxy). The linear precursor with protected side chains (1) was assembled by automated solid-phase peptide synthesis on 2-Chlorotrityl Chloride Resin (substitution of 0.536 mmol/g, 452 mg) using a 9-fluorenylmethoxycarbonyl (Fmoc) strategy with a synthesizer (LIBERTY 1-CEM) with microwave activation. After Fmoc titration (final loading of 0.321 mmol/g, 145 μmol, SPPS yield of 60%), the peptide was cleaved from the resin by treatment with a CH2Cl2/TFA solution [45 mL, 99/1 (v/v)] (2 × 3 min), and then the solvent was evaporated and the residue precipitated in cold Et2O to obtain an orange oil. Compound 1 was directly used for the next step without further purification. Analytical HPLC (Chromolith column, gradient from 50 to 100% B over 15 min): 90.6% purity, tR = 5.4 min. (−)ESI-MS: calcd for C87H122N14O28P2S [M − H]− m/z 1903.7, experimental [M − H]− m/z 1903.3 and [M − 2H]2− m/z 951.8. Cyclic Protected Peptide 2 c(Ser(PO3HBz)-Arg(Pbf)-Glu(tBu)-ProGly-Glu(tBu)-Trp(Boc)-Ser(PO3HBz)-Pro-Gly). Linear protected peptide 1 (54 μmol) was dissolved in a CH3CN/CH2Cl2 solvent (0.5 mM), and diisopropylethylamine (DIEA) (240 μL, 20 equiv) was added. Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (3 equiv) was added before microwave activation. The reaction was monitored using analytical RP-HPLC (Merck Chromolith). The solvent was concentrated and the residue precipitated in cold Et2O. The compound was dissolved in a CH3CN/H2O solvent (50/50), filtered on 0.45 μm PTFE, and purified by preparative RP-HPLC (Merck Chromolith) to yield pure protected cyclodecapeptide 2 (60 mg, 32 μmol, 59%) as a white solid after lyophilization. Analytical HPLC (Chromolith column, gradient from 50 to 100% B over 15 min): 92% purity, tR = 10.9 min. (−)ESIMS: calcd for C87H120N14O27P2S [M − H]− m/z 1885.7, experimental [M − H]− m/z 1885.6. Final Cyclic Peptide pS18 c(pSer-Arg-Glu-Pro-Gly-Glu-Trp-pSer-ProGly). Cyclic protected peptide 2 (60 mg, 32 μmol) was treated with a TFA/triisopropylsilane (TIS)/H2O solution [3 mL, 95/2.5/2.5 (v/ v)]. After being stirred for 3 h, the solution was evaporated under reduced pressure to afford a yellow oil, which was precipitated with cold Et2O to yield crude compound pS18 as a yellowish solid (48 mg). Pure cyclodecapeptide pS18 (17 mg, 12.5 μmol) was isolated after preparative RP-HPLC (Merck Chromolith) as a white solid after lyophilization (39% yield, 14% overall yield). Analytical HPLC (Chromolith column, gradient from 0 to 60% B over 30 min): 99.6% purity, tR = 12.8 min. (−)ESI-MS: calcd for C47H68N14O22P2 [M − H]− m/z 1241.4, experimental [M − H]− m/z 1241.4 and [M − 2H]2− m/z 620.3. Preparation of the Solutions for Physicochemical Studies. A stock uranyl solution (∼10 mM) was prepared from uranyl nitrate hexahydrate in 0.01 M nitric acid. The precise uranyl concentration was obtained by measuring the absorbance of an aliquot compared to that of an ICP uranium standard (1000 μg of U/mL in 2% HNO3) at λ = 415 nm. MES buffer (20 mM MES and 0.1 M NaCl) was prepared by dissolving solid 2-(N-morpholino)ethanesulfonic acid and sodium chloride in H2O and by adjusting the pH to 6.0 with KOH. HEPES buffer (20 mM HEPES and 0.1 M NaCl) was prepared by dissolving solid 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and sodium chloride in H2O and by adjusting the pH to 7.0 or 7.4 with KOH. A solution of IDA (∼0.1 M) was prepared by dissolving solid iminodiacetic acid in ultrapure water, and the precise concentration was determined by titration with a standard 0.1 M KOH solution. Buffer solutions containing IDA were prepared using previous solutions; the pH was then controlled and, if required, adjusted to 6.0, 7.0, or 7.4 with KOH. Peptide solutions were prepared freshly before use, and the precise peptide concentration was determined by recording a UV spectrum (λ280 = 5690 mol−1 cm−1 because of the presence of a Trp residue).

presence of four coordinating amino acids has been proven to be necessary for obtaining a high affinity for uranyl. The sequence pSDEpSDE, found in OPN, contains two phosphorylated residues, namely phosphoserines (pSer), known to bind uranyl strongly because of their phosphate moiety and several acidic residues, namely, glutamic (Glu) and aspartic (Asp) acids. Although OPN is an intrinsically disordered protein, it acquires some structure upon uranyl binding as shown by circular dichroism (CD) studies.24 The high affinity for uranyl is hence expected to be due to an optimal organization of four to six amino acid side chains from pSer, Glu, and Asp coordinated in the equatorial plane of the cation. Therefore, preorganized model peptides with appropriate residues and sequences derived from A1 may reflect more satisfactorily the uranyl-bound form of the protein than flexible linear sequences do. In this Article, we present the design of a series of cyclic preorganized peptides combining two phosphoserines and two glutamic acids as uranyl-binding residues to mimic the phosphorylated binding sites of OPN. Glutamic acid has been chosen as a preferred acidic residue because we demonstrated that it promotes a higher affinity for uranyl than aspartic acid because of both electronic and steric effects.33 The combination of complementary spectroscopic and analytical methods demonstrates that the sequence is a key parameter for the nature and structure of the uranyl complex. All the peptides demonstrate a particularly high affinity for uranyl in accordance with the high affinity of the whole OPN protein and also with the crucial role of the phosphorylated residues.

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EXPERIMENTAL SECTION

Synthesis of the Cyclic Peptides. This process is described for pS18 as an example. Characterization of the four compounds is given in the Supporting Information. Solvents for high-performance liquid 11558

DOI: 10.1021/acs.inorgchem.5b02249 Inorg. Chem. 2015, 54, 11557−11562

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Inorganic Chemistry ESI-MS Experiments. Peptide solutions (100 μM) were prepared in ammonium acetate buffer (20 mM, pH 7.0). The stock uranyl solution was added to prepare aliquots containing 0.5, 1, and 2 equiv of UO22+ per peptide. Mass spectra were recorded on a LXQ type Thermo Scientific spectrometer equipped with an electrospray ionization source and a linear trap detector. Solutions were injected into the spectrometer at a flow rate of 10 μL/min. The ionization voltage and capillary temperature were ∼2 kV and ∼250 °C, respectively. The source settings were the same (sheath gas, auxiliary gas, capillary voltage, and tube lens) for all the samples so comparable data could be obtained. CD Titrations. CD spectra were recorded at 25 °C on an Applied Photophysics Chirascan Spectrometer in a 1 cm path cell. The peptide concentration was ∼10−15 μM in water, and the pH was adjusted to 6.0 or 7.0 with KOH. All spectra were recorded from 320 to 190 nm with a datum interval of 1 nm, a time constant of 2 s, and a bandwidth of 1 nm, with three scans. CD spectra are reported in molar ellipticity ([Θ] in units of degrees square centimeters per decimole). [Θ] = Θobs/(10lc), where Θobs is the observed ellipticity in millidegrees, l the optical path length of the cell in centimeters, and c the peptide concentration in moles per liter. Fluorescence Titrations. Trp fluorescence quenching was followed by titration of a 10 μM peptide-buffered solution containing 0.1 mM IDA, with uranyl (from 0 to 4 equiv, aliquots of 0.1 equiv). The pH was measured at the beginning and end of the experiment to guarantee pH stability during titration. Spectra were recorded on an LS50B spectrofluorimeter connected to a computer equipped with FLWINLAB version 2.0. The measurements were performed at 298 K, in a 1 cm path cell. Trp fluorescence titrations were performed with 280 nm excitation (excitation slit of 3−13 nm). The emission slit was adjusted (3.5−4.5 nm) to avoid signal saturation. The conditional stability constants were extracted from the spectral data using SPECFIT, and taking into account the conditional stability constants of uranyl with the competing agent (IDA). The conditional stability constants of uranyl complexation by iminodiacetic acid (IDA) at pH 6.0, 7.0, and 7.4 were calculated from published global constants:34

1000, 5000, and 10000 equiv of Ca2+. Reverse titrations upon addition of a large excess of calcium first followed by uranyl give similar results, indicating that the thermodynamic equilibrium is reached.

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RESULTS Design and Synthesis of the Cyclic Peptides. The four phosphorylated peptides presented in Scheme 1 may be classified as cis- or trans-like, depending on the relative positions of the two pSer residues. Hence, pS18 and pS68 may be presented as “cis” compounds because the two phosphorus groups belong to the same β-turn in the pSerPro-Gly-pSer motif of pS18 or are separated by only one residue (Trp) in pS68. By contrast, pS16 and pS38 have two pSer residues in a trans-like relationship with respect to the elongated cycle. Indeed, the two pSer residues do not belong to the same β-turn and are separated by four amino acids, including a coordinating glutamate. The synthesis of the four phosphorus peptides was performed as reported previously by synthesizing the protected linear peptide on an acid-labile resin and subsequent cyclization in the liquid phase. The synthesis and characterization of the peptides are reported in the Experimental Section and Supporting Information. Mass Spectrometry of the Uranyl Complexes. The nature of the uranyl complexes was first investigated qualitatively by mass spectrometry (ESI-MS) in ammonium acetate buffer at pH 7.0 to be close to physiological conditions. ESI-MS clearly evidences tight uranyl complexes with the four peptides as seen in Figure 1 for pS16 and pS18. The free

UO2 2 + + IDA2 − = UO2 IDA; log β11pH 6 = 6.4; log β11pH 7 = 7.4; log β11pH 7.4 = 7.8 UO2 2 + + 2IDA2 − = UO2 (IDA)2 2 − ; log β12 pH 6 = 9.4; log β12 pH 7 = 11.4; log β12 pH 7.4 = 12.2 2UO2 2 + + 2IDA2 − + 2OH− = (UO2 )2 (IDA)2 (OH)2 2 − ; log β22 pH 6 = 15.8; log β22 pH 7 = 19.8; log β22 pH 7.4 = 21.4 Competition with Ca2+ Followed by Fluorescence. A 1 M stock solution of CaCl2 was prepared in water. Peptide solutions (∼5 μM) were prepared either at pH 6.0 (20 mM MES and 0.1 M NaCl) or at pH 7.4 (20 mM HEPES and 0.1 M NaCl). The Trp fluorescence was measured with excitation at 280 nm. The intensity value obtained for the free peptide was normalized to 1. The uranyl complex was formed by adding 2 equiv of the stock uranyl solution. Then aliquots of the stock calcium solution were added to obtain samples with 500,

Figure 1. (−)ESI-MS spectra of (top) pS16 and (bottom) pS18 with 1 and 2 equiv of UO22+ in ammonium acetate buffer (20 mM, pH 7.0). Asterisks denote sodium adducts. P stands for peptide. 11559

DOI: 10.1021/acs.inorgchem.5b02249 Inorg. Chem. 2015, 54, 11557−11562

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Inorganic Chemistry

organized (UO2)2·pS16 complex, with a nice β-sheet structure. Bridging phosphates may be proposed to explain such an amazing result. Dramatic differences are observed in the intensities of the CD bands centered at 207 nm in the uranyl complexes: pS18 (Θ ≈ 32000 deg cm2 dmol−1), pS68 (Θ ≈ 52000 deg cm2 dmol−1), pS38 (Θ ≈ −11000 deg cm2 dmol−1), and pS16 (Θ ≈ 142000 deg cm2 dmol−1). These intensities are correlated with the ability of the peptide to form a β-sheet, and therefore, we can conclude that sequences with pSer before a cyclic Pro residue are less prone to form the β-sheet structure, probably because of steric constraints that limit the orientation of the pSer side chain. The most striking effect is seen by comparing the two trans-like peptides pS16 and pS38. Indeed, in the latter compound, the two pSer residues are just before the two Pro residues, which may explain why the uranyl complex is poorly structured. By contrast, the related peptide pS16 with two pSer residues close to Gly is much more flexible and accommodates two uranyl ions in a well-defined structure. Fluorescence Titrations and Affinity Determination. The fluorescence of tryptophan (Trp) is an efficient probe for revealing uranyl complex formation and measuring stability, which is a key parameter for the design and understanding of uranyl chelation.33 Titrations of the peptides with uranyl demonstrate a very large or even total quenching of Trp emission at 350 nm (Figure S7). The experiments were conducted in the presence of a known quantity of iminodiacetic acid (IDA) to control uranyl speciation and in particular to avoid hydroxo species formation, which is crucial for reliable thermodynamic analyses. The stability constants of complexes formed with IDA are known;34 therefore, it is possible to deduce the conditional stability constant of the uranyl complexes with peptides at a given pH. This method has been validated in a previous detailed report with polyacidic peptides at pH 6.33 The conditional constants (KC) measured in this work are very sensitive to pH because the diphosphorylated peptides are quite basic. Indeed, the usual pKa value of the phosphate group in polypeptides is ∼5.7. However, it is expected that the pKa value of the second phosphate group is >5.7. Results obtained from fluorescence titrations in the presence of IDA at three pH values are reported in Table 1. As expected, the KC values increase significantly with an increase in pH, and this evolution can be satisfactorily interpreted with phosphate pKa values of 5.7−7 and 7.5−8.5. Hence, the diphosphorylated peptides show moderate affinity for uranyl at acidic pH because one of the phosphate functions is protonated. Uranyl detection via Trp

peptide is a minor species detected in samples prepared with equimolar UO22+ (Figure 1, left). The 1/1 complex is nicely evidenced as a dianion in all the MS spectra (m/z 754.3 [UO2P − 4H]2−), demonstrating the formation of a mononuclear uranyl complex as previously observed with peptide A1. The bimetallic complex is detected in an excess of uranyl {m/z 888.3 [(UO2)2P − 6H]2−} and remains a very minor species except for pS16 (Figure 1, top). The latter compound shows a specific behavior with the bimetallic species being the major ion detected in the MS spectra in excess of uranyl. Circular Dichroism. CD titrations are informative in terms of the structural changes experienced by the peptide scaffold upon metal complexation. As an exemple, Figure 2 shows the

Figure 2. CD titration of (top) pS16 and (bottom) pS18 at ∼10 μM with UO22+ (0−2.5 equiv; 0.25 equiv aliquots) at pH 7.0.

titrations of peptides pS16 and pS18 with UO22+ followed by CD in the far-UV region, which is indicative of the backbone structure. The apo peptides show the characteristic features of unstructured peptide backbones at pH 7, which is related to the strong charge repulsions between the deprotonated phosphates and carboxylate groups that prevent the formation of the βsheet structure. All the peptides show the buildup of the band characteristic of β-sheet formation at 207 nm upon addition of uranyl (Figure 2 and Figure S6). The formation of predominant mononuclear UO2·P complexes, detected in the ESI-MS experiments, is confirmed by an end point at 1 equiv of UO22+ in the CD titrations with compounds pS18, pS68, and pS38. By contrast, the CD band at 207 nm evolves until 2 equiv of UO22+ are added for pS16, confirming the peculiar behavior of this “trans” peptide. The latter band is very intense, which indicates that the two uranyl ions are coordinated in a highly

Table 1. Conditional Stability Constant Values (log KC, where KC = [ML]/[M][L]) for the UO2P Complex at Three Different pH Valuesa peptide

log KC (pH 6)

log KC (pH 7)

log KC (pH 7.4)

A133 pS18 pS68 pS16

8.2(1) 8.6(1) 8.2(1) 8.7(2)

not determined 9.7(2) 10.0(1) 10.1(2)

pS38

8.8(2)

9.8(1)

not determined 10.1(2) 10.3(1) 10.3(1) 9.3(2)b 10.0(2)

a

Conditional constant values in buffer solutions (20 mM) at pH 6.0 (MES), 7.0 (HEPES), and 7.4 (HEPES) containing 0.1 M NaCl and IDA as a competitor of known affinity (100 μM).34 bLog K2, where K2 = [M2L]/[ML][M]. 11560

DOI: 10.1021/acs.inorgchem.5b02249 Inorg. Chem. 2015, 54, 11557−11562

Inorganic Chemistry

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DISCUSSION Preorganized peptide scaffolds such as A1 (Scheme 1) with four acidic amino acids were recently validated as efficient uranyl-binding peptides. These compounds were derived here to insert very efficient phosphorus uranyl-binding groups, namely phosphoserines. A series of cyclic derivatives were developed to mimic binding sites thought to effectively chelate uranyl in phosphorylated proteins such as osteopontin thanks to two acidic residues and two phosphoserines. Complementary physicochemical techniques demonstrate that the sequence is a critical factor for the type and structure of the uranyl complex formed. Mononuclear species are evidenced with three of the compounds with a stronger ability to adopt a β-sheet conformation when the pSer residues are placed prior to unhindered Gly residues and hence in a poorly constrained environment. In contrast, peptide pS16 with two pSer residues in a trans-like relationship with respect to the peptide backbone is able to chelate two uranyl ions in a wellstructured β-sheet, suggesting bridging phosphate groups that maintain the bimetallic core. Phosphoserine residues significantly enhance the stability of uranyl peptide complexes. Indeed, the conditional stability constants at physiological pH are 2 orders of magnitude higher that those measured with the related polyacidic compound A1. This emphasizes the crucial contribution of the phosphoserine amino acid in uranyl coordination. Surprisingly, the position of the two phosphoserines and the ability of the peptide scaffold to adopt a β-sheet structure have an only moderate influence on the uranyl complex stability. Therefore, the coordination of the phosphate groups can be considered as the main driving force for the stability of the uranyl complexes within this series of cyclic diphosphoserine peptides. Unfortunately, a rigorous comparison of the affinity of the model peptides with that of the OPN protein is impossible because the conditional stability constants of the related uranyl complexes have been measured under different experimental conditions (pH, buffer, competitor, etc.). Importantly, these preorganized peptides may reflect the structure of the uranyl-bound form of OPN, for which flexibility allows an optimal arrangement of the coordinating residues in the equatorial plane of the dioxo cation. Because little structural information is currently available, these model compounds are crucial to improving our understanding of the binding of uranyl to proteins related to toxicity. Finally, these models are demonstrated to very efficiently chelate uranyl even in the presence of calcium. Such motifs are hence fully relevant for uranyl binding in vivo; other sequences are currently being developed to predict the most efficient uranyl-binding sites in phosphorylated proteins.

quenching in a peptide resembling pS38 was recently proposed at pH 6.35 The data presented here indicate that under these acidic conditions, the stabilities of the uranyl complexes formed with diphosphorylated peptides are not optimal and polyacidic cyclic peptides like A1 may be better alternatives with similar affinities for uranyl and very large fluorescence quenching. By contrast, at physiological pH, the diphosphorylated peptides described here become very efficient for uranyl binding with affinity constants of >1010, because the two phosphate groups are deprotonated and are consequently fully playing their metal-chelating role. Competition with Calcium. In vivo, OPN is known to tightly bind the physiological ion Ca2+,25 and it was recently shown that this high affinity is independent of the phosphorylation status of the protein.36 Therefore, uranyl chelation was studied in the presence of Ca2+. As seen in Figure 3, Ca2+ alone does not significantly quench the peptide

Figure 3. Trp normalized fluorescence intensities at 350 nm (λexc = 280 nm) of peptide solutions (5 μM) at (top) pH 6.0 or (bottom) pH 7.4. The uranyl complex was formed by adding 2 equiv of UO22+ to the peptides. Then 500, 1000, 5000, or 10000 equiv of Ca2+ per peptide was added to the latter complex. The fluorescence of the peptide with 10000 equiv of Ca2+ is shown for comparison.

fluorescence, and therefore, the competition between UO22+ and Ca2+ can be evidenced by an increase in Trp fluorescence upon addition of Ca2+. The fluorescence intensities of the UO22+ complexes are shown in Figure 3 as a function of added Ca2+. No significant variation is detected in the experiments conducted at pH 6 even in a 10000-fold excess of Ca2+ (Figure 3, top). By contrast, the competition between the two cations is much more effective at physiological pH and the fluorescence increases significantly above 5000 equiv of Ca2+ added. However, Figure 3 (bottom) shows that 50−70% of the uranyl complex is still formed in the presence of 10000 equiv of Ca2+, which indicates binding constants 4 orders of magnitude higher for UO22+ than for Ca2+. Hence, such binding sites are fully realistic for uranyl chelation in vivo, with the calcium concentration in the millimolar range.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b02249. Characterization of peptides and supplementary spectroscopic figures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 11561

DOI: 10.1021/acs.inorgchem.5b02249 Inorg. Chem. 2015, 54, 11557−11562

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Inorganic Chemistry Notes

(28) Dumy, P.; Eggleston, I. M.; Esposito, G.; Nicula, S.; Mutter, M. Biopolymers 1996, 39, 297. (29) Bonnet, C. S.; Fries, P. H.; Crouzy, S.; Sénèque, O.; Cisnetti, F.; Boturyn, D.; Dumy, P.; Delangle, P. Chem. - Eur. J. 2009, 15, 7083. (30) Pujol, A. M.; Cuillel, M.; Renaudet, O.; Lebrun, C.; Charbonnier, P.; Cassio, D.; Gateau, C.; Dumy, P.; Mintz, E.; Delangle, P. J. Am. Chem. Soc. 2011, 133, 286. (31) Fragoso, A.; Lamosa, P.; Delgado, R.; Iranzo, O. Chem. - Eur. J. 2013, 19, 2076. (32) Fragoso, A.; Delgado, R.; Iranzo, O. Dalton Trans. 2013, 42, 6182. (33) Lebrun, C.; Starck, M.; Gathu, V.; Chenavier, Y.; Delangle, P. Chem. - Eur. J. 2014, 20, 16566. (34) Jiang, J.; Sarsfield, M. J.; Renshaw, J. C.; Livens, F. R.; Collison, D.; Charnock, J. M.; Helliwell, M.; Eccles, H. Inorg. Chem. 2002, 41, 2799. (35) Yang, C. T.; Han, J.; Gu, M.; Liu, J.; Li, Y.; Huang, Z.; Yu, H. Z.; Hu, S.; Wang, X. Chem. Commun. 2015, 51, 11769. (36) Klaning, E.; Christensen, B.; Sørensen, E. S.; Vorup-Jensen, T.; Jensen, J. K. Bone 2014, 66, 90.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the “Programme Transversal Toxicologie du CEA”, the NRBC-E program, and the Labex ARCANE (Grant ANR-11-LABX-0003-01).

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REFERENCES

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DOI: 10.1021/acs.inorgchem.5b02249 Inorg. Chem. 2015, 54, 11557−11562