Rotational modes of Ca2+-liganded calmodulin, as determined by time-domain fluorescence

The time decay of fluorescence anisotropy was monitored as a function of pH and temperature for complexes of 2,6-toluidinylnaphthalenesulfonate with calmodulin, with its proteolytic fragments, and with the 1:1 complex of calmodulin and melittin. For all the conditions examined the anisotropy decay of native calmodulin involved at least two rotational modes. These corresponded to a short correlation time of 2-3 ns, reflecting a localized motion in the vicinity of the binding site and a longer correlation time which arises from the rotation of a major portion of the molecule. The relative amplitudes of the two rotational modes were dependent upon temperature in the range 11-40 degrees C, the contribution of the more rapid mode increasing with temperature. The maximum immobilization of the probe occurred at pH 5.0 and 12 degrees C. While these results indicate the presence of internal rotations in Ca2+-liganded calmodulin, the magnitude of the longer correlation time is consistent with the crystallographic structure.