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

Developing chelators that combine high affinity and selectivity for lanthanides and/or actinides is paramount for numerous industries, including rare earths mining, nuclear waste management, and cancer medicine. In particular, achieving selectivity between actinides and lanthanides is notoriously difficult. The protein lanmodulin (LanM) is one of Nature's most selective chelators for trivalent actinides and lanthanides. However, mechanistic understanding of LanM's affinity and selectivity for f-elements remains limited. In order to decipher, and possibly improve, the features of LanM's metal-binding sites that contribute to this actinide/lanthanide selectivity, we characterized five LanM variants, substituting the aspartate residue at the 9 th position of each metal-binding site with asparagine, histidine, alanine, methionine, and selenomethionine. Spectroscopic measurements with lanthanides (Nd 3+ and Eu 3+ ) and actinides ( 243 Am 3+ and 248 Cm 3+ ) reveal that, contrary to the behavior of small chelator complexes, metal-coordinated water molecules enhance LanM's affinity for f-elements and pH-stability of its complexes. Furthermore, the results show that the native aspartate does not coordinate the metal directly but rather hydrogen bonds to coordinated solvent. By tuning this first-sphere/second-sphere interaction, the asparagine variant nearly doubles LanM's selectivity for actinides versus lanthanides. This study not only clarifies the essential role of coordinated solvent for LanM's physiological function and separation applications, but it also demonstrates that LanM's preference for actinides over lanthanides can be further improved. More broadly, it demonstrates how biomolecular scaffolds possess an expanded repertoire of tunable interactions compared to most small-molecule ligands - providing an avenue for high-performance LanM-based actinide/lanthanide separation methods and bio-engineered chelators optimized for specific medical isotopes.