Characterizing the Lanthanide‐Binding Tag Grafted at Calmodulin Site 1: Affinity, Selectivity, and Coordination Properties

Lanthanide‐binding tag (LBT) optimized for protein labeling was engineered into calmodulin by inserting a variant sequence (W 7 Y 8 → Y 7 I 8 ) at Site 1 of the N‐terminal domain while inactivating Site 2, and the resulting CaMLBT was examined for its Ln‐binding properties. CaMLBT forms 1:1 complexes with all lanthanides. Fluorescence spectroscopy and CE‐ICP‐MS revealed dissociation constants ranging from sub‐nanomolar for La (Kd = 437 ± 259 pM) to the low picomolar range for Tb–Lu, reaching Kd = 1.1 ± 0.4 pM for Lu at pH 6. This corresponds to a ∼1000‐fold affinity increase over the original LBT, approaching the stabilities of lanmodulins. In contrast to lanmodulins, however, Ln–CaMLBT complex stability increases with decreasing Ln(III) ionic radius, consistent with trends reported for LBT. ATR‐FTIR spectroscopy indicates that the enhanced stability arises from changes in coordination number and ligand properties: Glu 9 would evolve from tight bidentate (La to Pr) to more asymmetric or weaker (Yb and Lu) coordination, while Asp 3 and Asp 5 evolve from pseudo‐bridging to strong monodentate ligands. Notably, no evidence for water ligation was found for early lanthanides. CaMLBT emerges as a highly stable and versatile scaffold to investigate structural factors underlying lanthanide selectivity and complex stability.