[en] In spite of an increasing number of publications in recent years, information regarding the mechanism of uranium interaction with proteins at the molecular level is limited and few quantitative studies have investigated the binding properties of uranyl with proteins or peptides. It is thus of great interest to better characterize these interactions, and to analyze structural factors governing uranyl binding and thermodynamic stabilization in proteins. Research in this direction will benefit our understanding of the molecular factors governing uranyl toxicity and speciation in cells and will also aid in developing new molecules for selectively binding uranium that could be used for uranium bioremediation purposes. Uranyl coordination properties have similarities with those of calcium, i.e electrostatic interactions preferentially with hard donor oxygen ligands and pentagonal bipyramidal structures. The EF-hand structural motif is the most prevalent Ca2^+-binding site in proteins and is very appealing to analyse uranyl binding properties and develop affine and specific uranyl binding sites for biotechnology approaches. We selected the recombinant N-terminal domain of calmodulin from A. thaliana as a structured template that contains two EF-hand motifs (site I and site II) to analyze its uranyl binding properties and to engineer peptide variants with increased uranyl affinity and specificity. We showed that both site I and site II bind uranyl, with a dissociation constant in the nano-molar range for site I (K_d ≅ 25 nM at pH 6). Using in vitro phosphorylation of a threonine located in the uranyl binding loop, we measured how adding a phosphoryl group affects the calcium and uranium binding affinities. The phosphorylated peptide exhibited a very large affinity for uranyl at pH 7, with a dissociation constant in the sub-nano-molar range K_d = 0.25 ±0.06 nM, and FTIR analysis demonstrated that the phosphoryl group plays a determining role in uranyl binding affinity. We then combined site directed mutagenesis and a modeling approach based on molecular dynamics to increase both the affinity and specificity of the EF-hand motif for uranyl. We could thus obtain a uranyl binding motif with a dissociation constant of 200 pM at pH 6 without phosphorylation and a uranyl/calcium specificity of 10⁷. These results may open new routes for environmental monitoring of uranium based on the use of engineered peptides with high uranyl affinity and specificity. Document available in abstract form only. (authors)