Reversing Lanmodulin's Metal‐Binding Sequence in Short Peptides Surprisingly Increases the Lanthanide Affinity

Abstract Rare earth elements (REEs) are essential for a clean energy future, high tech, and medicine. For these applications the chemically similar elements need to be tediously separated. Recent discoveries that specific lanthanide‐binding proteins such as lanmodulin (LanM) exist in nature have prompted the development of bio‐inspired separation methods for REEs. Peptides hold great potential for tuning binding sites in various applications as they are easily synthesised, modifiable, and can be immobilised. Here we use the EF‐hand binding site sequences of LanM, the naturally lanthanide‐binding EF‐hand protein from Methylorubrum extorquens AM1, as a blueprint for peptides with potential applications in REE recycling. We show with time‐resolved laser‐induced fluorescence spectroscopy (TRLFS), isothermal titration calorimetry (ITC), and nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics (MD) simulations and circular dichroism (CD) spectroscopy the surprising result that reversing the natural sequence of LanM's metal‐binding loops leads to an increased binding affinity of about one order of magnitude for three out of four natural sequences. Furthermore, we were able to identify structural features responsible for the affinity boost and were able to obtain – only by exchanging one amino acid – a linear uncapped 12‐amino‐acid peptide with a 150 nM affinity for lanthanides.