Although the 3D structure of the Ca2+-bound CaM (Ca2+/CaM) complex with the antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), has been resolved, the dynamic changes in Ca2+/CaM structure upon interaction with W-7 are still unknown. We investigated time- and temperature-dependent dynamic changes in Ca2+/CaM interaction with W-7 in physiological conditions using one- and two-dimensional Fourier-transformed infrared spectroscopy (2D-IR). We observed changes in the alpha-helix secondary structure of Ca2+/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 125 s at 25 degrees C, Ca2+/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 Ca2+/CaM complex failed to do so when Ca2+/CaM was complexed with W-7. We concluded that W-7 induced stepwise conformational changes in Ca2+/CaM that resulted in a rigidification of the complex and its inability to interact with target proteins and/or polypeptides. (C) 2012 Elsevier Inc. All rights reserved.