Although the 3D structure of the Ca(2+)-bound CaM (Ca(2+)/CaM) complex with the antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), has been resolved, the dynamic changes in Ca(2+)/CaM structure upon interaction with W-7 are still unknown. We investigated time- and temperature-dependent dynamic changes in Ca(2+)/CaM interaction with W-7 in physiological conditions using one- and two-dimensional Fourier-transformed infrared spectroscopy (2D-IR). We observed changes in the α-helix secondary structure of Ca(2+)/CaM when complexed with W-7 at a molar ratio of 1:2, but not at higher molar ratios (between 1:2 and 1:5). Kinetic studies revealed that, during the initial 125s at 25°C, Ca(2+)/CaM underwent formation of secondary coil and turn structures upon binding to W-7. Variations in temperature that induced significant changes in the structure of the Ca(2+)/CaM complex failed to do so when Ca(2+)/CaM was complexed with W-7. We concluded that W-7 induced stepwise conformational changes in Ca(2+)/CaM that resulted in a rigidification of the complex and its inability to interact with target proteins and/or polypeptides.