Biofunctionality of Calmodulin Immobilized on Gold Surface Studied by Surface-Enhanced Infrared Absorption Spectroscopy: Ca2+-Induced Conformational Change and Binding to a Target Peptide

Calcium ion (Ca 2+ )-induced functional expression of calmodulin (CaM), which is a signal transmitter in living cell, is known to be associated with its conformation change; however, it is difficult to correlate the conformational change and functional expression induced by Ca 2+ reversibly as it is not easy to change the Ca 2+ concentration while keeping the CaM concentration in solution. This can be done if CaM is immobilized on a solid surface while maintaining its function intact. Here CaM is immobilized on a self-assembled monolayer (SAM) of Ni-nitrilotriacetic acid (NTA) on a Au surface by histidine tag (His-tag) method. The conformational change of the immobilized CaM induced by the Ca 2+ concentration change in solution was investigated using surface-enhanced infrared absorption spectroscopy (SEIRAS). The Ca 2+ -induced conformational change of CaM immobilized on the Au surface was followed by monitoring IR intensities at 1550 and 1645 cm –1, which originated from the Ca 2+ binding site (EF hand) in CaM and the central helix portion in CaM, respectively. The conformation of CaM was confirmed to be changed reversibly when the concentration of Ca 2+ was increased and decreased between 10 –5 and 10 –3 M in a phosphate-buffered D 2 O solution. The isotherm for immobilized CaM with respect to the Ca 2+ concentration is in good agreement with that in solution, which confirms that the activity of CaM is maintained when immobilized on the surface. The interaction between CaM and a target peptide, mastoparan (MP), was also investigated using SEIRAS. Although MP was reversibly bound to and released from the CaM immobilized on the Au surface depending on the presence and absence of Ca 2+, respectively, the dissociation constant of CaM immobilized on Au surface with MP was determined from the MP concentration dependence of attachment of MP as two orders of magnitude larger than that of CaM in homogeneous environment, possibly due to the difficulty for MP to access the binding site of CaM, which is immobilized on the surface with high order.