Probing Lanmodulin’s Lanthanide Recognition via Sensitized Luminescence Yields a Platform for Quantification of Terbium in Acid Mine Drainage

Abstract

Lanmodulin is the first natural, selective macrochelator for f elementsa protein that binds lanthanides with picomolar affinity at 3 EF hands, motifs that instead bind calcium in most other proteins. Here, we use sensitized terbium luminescence to probe the mechanism of lanthanide recognition by this protein as well as to develop a terbium-specific biosensor that can be applied directly in environmental samples. By incorporating tryptophan residues into specific EF hands, we infer the order of metal binding of these three sites. Despite lanmodulin’s remarkable lanthanide binding properties, its coordination of approximately two solvent molecules per site (by luminescence lifetime) and metal dissociation kinetics (k off = 0.02–0.05 s–1, by stopped-flow fluorescence) are revealed to be rather ordinary among EF hands; what sets lanmodulin apart is that metal association is nearly diffusion limited (k on ≈ 109 M–1 s–1). Finally, we show that Trp-substituted lanmodulin can quantify 3 ppb (18 nM) terbium directly in acid mine drainage at pH 3.2 in the presence of a 100-fold excess of other rare earths and a 100 000-fold excess of other metals using a plate reader. These studies not only yield insight into lanmodulin’s mechanism of lanthanide recognition and the structures of its metal binding sites but also show that this protein’s unique combination of affinity and selectivity outperforms synthetic luminescence-based sensors, opening the door to rapid and inexpensive methods for selective sensing of individual lanthanides in the environment and in-line monitoring in industrial operations.

Engineering lanmodulin's selectivity for actinides over lanthanides by controlling solvent coordination and second-sphere interactions

“…Based on these equations, Cm 3+ and Eu 3+ are surrounded by 2.3 and 2.0 water molecules, respectively, within the binding sites of WT LanM ( Table 2 ). This is consistent with independent measurements recently performed 52 with Tb 3+ and LanM containing tryptophan residues as luminescence sensitizers. Based on the Kimura et al 80 and Horrocks et al 81 equations, the 3D 9 N variant complexes of Cm 3+ and Eu 3+ have comparable numbers of water molecules relative to their WT counterparts, whereas the 3D 9 A, 3D 9 M, 3D 9 SeMet, and 3D 9 H complexes have slightly fewer coordinated water molecules.…”

Bridging Hydrometallurgy and Biochemistry: A Protein-Based Process for Recovery and Separation of Rare Earth Elements

“…Interestingly, in solution, LanM binds 3 equiv of REEs (Figure S1), suggesting that one metal site is destabilized or inaccessible upon immobilization, or in the column format. We suggest that this observation reflects the greater lability of metal ions bound to EF hand 1, especially at pH ≤ 5, 36 which may lead this site to not be stably occupied under flow-through conditions. The lability of EF hand 1 may also explain the requirement that LanM be added at a 1:2 (REE:protein) rather than 1:3 stoichiometry to achieve quantitative REE extraction in prior experiments with industrial feedstock leachates.…”

Repeated Recovery of Rare Earth Elements Using a Highly Selective and Thermo‐Responsive Genetically Encoded Polypeptide

“…They are usually characterized by higher concentrations of various non‐REE ions and very low REE content. [ 35 ] LanM was used to monitor the REE content in acid mine drainage. [ 35 ] Furthermore, the real world samples are considered as resources of REEs.…”

“…[ 35 ] LanM was used to monitor the REE content in acid mine drainage. [ 35 ] Furthermore, the real world samples are considered as resources of REEs. In particular, iron metallurgy slags are abundant industrial wastes and potentially valuable resources for REEs.…”