A Simple Fluorescence Affinity Assay to Decipher Uranyl-Binding to Native Proteins.

Determining the affinity of proteins for uranyl is key to understand the toxicity of this cation and to further develop decorporation strategies. However, usual techniques to achieve that goal often require specific equipment and expertise. Here, we propose a simple, efficient, fluorescence-based method to assess the affinity of proteins and peptides for uranyl, at equilibrium and in buffered solution. We first designed and characterized an original uranyl-binding fluorescent probe. We then built a reference scale for uranyl affinity in solution, relying on signal quenching of our fluorescent probe in presence of high-affinity uranyl-binding peptides. We finally validated our approach by re-evaluating the uranyl-binding affinity of four native proteins. We envision that this tool will facilitate the reliable and reproducible assessment of affinities of peptides and proteins for uranyl.

In vitro assessment of cobalt oxide particle dissolution in simulated lung fluids for identification of new decorporating agents.

Inhalation of 60Co3O4 particles may occur at the work place in nuclear industry. Their low solubility may result in chronic lung exposure to γ rays. Our strategy for an improved therapeutic approach is to enhance particle dissolution to facilitate cobalt excretion, as the dissolved fraction is rapidly eliminated, mainly in urine. In vitro dissolution of Co3O4 particles was assessed with two complementary assays in lung fluid surrogates to mimic a pulmonary contamination scenario. Twenty-one molecules and eleven combinations were selected through an extensive search in the literature, based on dissolution studies of other metal oxides (Fe, Mn, Cu) and tested for dissolution enhancement of cobalt particles after 1-28 days of incubation. DTPA, the recommended treatment following cobalt contamination did not enhance 60Co3O4 particles dissolution when used alone. However, by combining molecules with different properties, such as redox potential and chelating ability, we greatly improved the efficacy of each drug used alone, leading for the highest efficacy, to a 2.7 fold increased dissolution as compared to controls. These results suggest that destabilization of the particle surface is an important initiating event for a good efficacy of chelating drugs, and open new perspectives for the identification of new therapeutic strategies.

Cyclic Phosphopeptides to Rationalize the Role of Phosphoamino Acids in Uranyl Binding to Biological Targets.

The specific molecular interactions responsible for uranium toxicity are not yet understood. The uranyl binding sites in high-affinity target proteins have not been identified yet and the involvement of phosphoamino acids is still an important question. Short cyclic peptide sequences, with three glutamic acids and one phosphoamino acid, are used as simple models to mimic metal binding sites in phosphoproteins and to help understand the mechanisms involved in uranium toxicity. A combination of peptide design and synthesis, analytical chemistry, extended X-ray absorption fine structure (EXAFS) spectroscopy, and DFT calculations demonstrates the involvement of the phosphate group in the uranyl coordination sphere together with the three carboxylates of the glutamate moieties. The affinity constants measured with a reliable analytical competitive approach at physiological pH are significantly enhanced owing to the presence of the phosphorous moiety. These findings corroborate the importance of phosphoamino acids in uranyl binding in proteins and the relevance of considering phosphoproteins as potential uranyl targets in vivo.

Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

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 ß-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 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity.

Pseudo-peptides based on methyl cysteine or methionine inspired from Mets motifs found in the copper transporter Ctr1.

Most proteins involved in Cu homeostasis bind to intracellular Cu(I) in stable Cu(S-Cys)x environments, thanks to well-conserved cysteine-rich sequences. Similarly, the Cu(I) transport protein Ctr1, responsible for copper acquisition, binds Cu(I) in Cu(S-Met)3 environments in conserved methionine-rich MXMXXM sequences, referred as Mets motifs. Pseudo-peptides based on a nitrilotriacetic acid scaffold and functionalized with three amino acids bearing thioether side chains, either methyl cysteine in T(1) or methionine in T(2), were synthesized as mimics of the Mets sequences found in Ctr1. These two ligands were obtained with good overall yields from commercial amino acids and demonstrate efficient chelating ability for Cu(I). Only one species, the mononuclear [CuT(1,2)](+) complex, was evidenced by electrospray ionization-mass spectroscopy (ESI-MS) and the circular dichroism signature obtained for the most constrained CuT(1) complex having the shortest side chains showed reorganization of the pseudo-peptide scaffold upon Cu(I) complexation. Considering that thioether functions are neutral sulfur donors, the stability constants measured by competition with ferrozine are quite large: log K ≈ 10.2-10.3. The CuT(1,2) complexes are significantly more stable that those formed with linear peptides, mimicking isolated Mets motifs MXMXXM of the Cu transport protein Ctr1 (log K ≈ 5-6). This may be attributed to the preorganized pseudo-peptide scaffold, which arranges the three neutral sulfur donors toward the metal center. Such moderate affinity Cu(I) chelators are interesting for applications in chelation therapy, for instance, to induce minimum disturbance of Cu homeostasis in Wilson's disease treatments.

Engineering short peptide sequences for uranyl binding.

Peptides are interesting tools to rationalize uranyl-protein interactions, which are relevant to uranium toxicity in vivo. Structured cyclic peptide scaffolds were chosen as promising candidates to coordinate uranyl thanks to four amino acid side chains pre-oriented towards the dioxo cation equatorial plane. The binding of uranyl by a series of decapeptides has been investigated with complementary analytical and spectroscopic methods to determine the key parameters for the formation of stable uranyl-peptide complexes. The molar ellipticity of the uranyl complex at 195 nm is directly correlated to its stability, which demonstrates that the ß-sheet structure is optimal for high stability in the peptide series. Cyclodecapeptides with four glutamate residues exhibit the highest affinities for uranyl with log KC =8.0-8.4 and, therefore, appear as good starting points for the design of high-affinity uranyl-chelating peptides.

A series of tripodal cysteine derivatives as water-soluble chelators that are highly selective for copper(I).

A series of tripodal ligands derived from nitrilotriacetic acid and extended by three converging, metal-binding, cysteine chains was synthesised. Their ability to bind soft metal ions thanks to their three thiolate functions was investigated by means of complementary analytical and spectroscopic methods. Three ligands that differ by the nature of the carbonyl group next to the coordinating thiolate functions were studied: L(1) (ester), L(2) (amide) and L(3) (carboxylate). The negatively charged derivative L(3), which bears three carboxylate functions close to the metal binding site, gives polynuclear copper(I) complexes of low stability. In contrast, the ester and amide derivatives L(1) and L(2) are efficient Cu(I) chelators with very high affinities, close to that reported for the metal-sequestering metallothioneins (log K≈19). Interestingly, these two ligands form mononuclear copper complexes with a unique MS(3) coordination in water solution. An intramolecular hydrogen-bond network involving the amide functions in the upper cavity of the tripodal ligands stabilises these mononuclear complexes and was evidenced by the very low chemical-shift temperature coefficient of the secondary amide protons. Moreover, L(1) and L(2) display large selectivities for the targeted metal ion that is, Cu(I), with respect to bioavailable Zn(II). Therefore the two sulfur-based tripods L(1) and L(2) are of potential interest for intracellular copper detoxication in vivo, without altering the homeostasis of the essential metal ion Zn(II).

Model peptides based on the binding loop of the copper metallochaperone Atx1: selectivity of the consensus sequence MxCxxC for metal ions Hg(II), Cu(I), Cd(II), Pb(II), and Zn(II).

The amino acid sequence MxCxxC is conserved in many soft-metal transporters that are involved in the control of the intracellular concentration of ions such as Cu(I), Hg(II), Zn(II), Cd(II), and Pb(II). A relevant task is thus the selectivity of the motif MxCxxC for these different metal ions. To analyze the coordination properties and the selectivity of this consensus sequence, we have designed two model peptides that mimic the binding loop of the copper chaperone Atx1: the cyclic peptide P(C) c(GMTCSGCSRP) and its linear analogue P(L) (Ac-MTCSGCSRPG-NH2). By using complementary analytical and spectroscopic methods, we have demonstrated that 1:1 complexes are obtained with Cu(I) and Hg(II), whereas 1:1 and 1:2 (M:P) species are successively formed with Zn(II), Cd(II), and Pb(II). The complexation properties of the cyclic and linear peptides are very close, but the cyclic compound provides systematically higher affinity constants than its unstructured analogue. The introduction of a xPGx motif that forms a type II beta turn in P(C) induces a preorganization of the binding loop of the peptide that enhances the stabilities of the complexes (up to 2 orders of magnitude difference for the Hg complexes). The affinity constants were measured in the absence of any reducing agent that would compete with the peptides and range in the order Hg(II) > Cu(I) >> Cd(II) > Pb(II) > Zn(II). This sequence is thus highly selective for Cu(I) compared to the essential ion Zn(II) that could compete in vivo or compared to the toxic ions Cd(II) and Pb(II). Only Hg(II) may be an efficient competitor of Cu(I) for binding to the MxCxxC motif in metalloproteins.