In Situ IRRAS Study of Myoglobin Binding to Metal-Chelating Nitrilotriacetate-Functionalized Binary Monolayers at the Air–Water Interface

Molecular-level understanding of protein binding to metal-chelating nitrilotriacetate (NTA)-functionalized biological surfaces remains poorly supported by direct spectroscopic evidence. Herein, in situ infrared reflection absorption spectroscopy (IRRAS) was employed to investigate the binding of native myoglobin (Mb, with five surface-exposed histidine residues in its native state) to binary monolayers consisting of NTA-functionalized amide-linked dioleoyl lipid (DOGS-NTA) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at the air–water interface in the presence of Cu2+ or Ni2+. No Mb binding to the DOGS-NTA monolayer occurred without divalent metal ions, nor was Mb binding observed for the pure DOPC monolayer even with Cu2+ or Ni2+. Robust Mb binding to the binary monolayers was achieved via NTA–metal–histidine ternary coordination. The amide I band intensities of specifically bound Mb at a surface pressure of 30 mN/m were approximately 4-fold higher in the Cu2+ case than those in the Ni2+ case, indicating markedly enhanced protein accumulation at the interface. The higher histidine affinity, faster ligand exchange, and Jahn–Teller geometric flexibility of Cu2+ accounted for the enhanced protein binding observed at the Cu2+-chelating interface. This molecular-level understanding of metal-ion-dependent protein binding mechanisms offers guidelines for the rational design of affinity biosensors, controlled-release delivery systems, and biomimetic interfaces.