Preparation of a fluorescent-labeled derivative of calmodulin which retains its affinity for calmodulin binding proteins

Abstract

Calmodulin was derivatized with 5-[[[(iodoacetyl)amino]ethyl]amino]-1-naphthalenesulfonic acid to fluorescently label the protein. This derivative (AEDANS.CaM) stimulated the Ca2+-sensitive cyclic nucleotide phosphodiesterase and formed Ca2+-dependent complexes with troponin I and the phosphodiesterase. Association between AEDANS.CaM and these proteins was directly monitored by changes in fluorescence anisotropy. The dissociation constants for the AEDANS.CaM--troponin I and AEDANS.CaM--phosphodiesterase complexes were 60 nM and 4 nM, respectively. This fluorescent derivative of calmodulin appears suitable for direct monitoring of the complexes between calmodulin and calmodulin binding proteins. Rotational diffusion of AEDANS.CaM was also measured with fluorescence anisotropy. These measurements indicated that the shape of calmodulin in solution is best approximated by a prolate ellipsoid.