Directly observed hydrogen bonds at calcium-binding-sites of calmodulin in solution relate to affinity of the calcium-binding

Molecular tuning to calcium-binding in the EF-hand motif of holo-calmodulin was studied in solution by NMR ^h3J_NC′ H-bond couplings. In the N-terminus lobe of holo-calmodulin, the glutamate crucial for Ca²⁺ coordination has network of H-bonds weaker than inferred from the X-ray crystal structure. This glutamate at position 12 appears shifted away from the Ca²⁺ preferred coordination, which can explain the lower affinity of the calcium-binding to the N-terminus with respect to C-terminus EF hands.