Interaction of a fluorescent N-dansylaziridine derivative of troponin I with calmodulin in the absence and presence of calcium

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Abstract
Rabbit skeletal muscle troponin I was covalently labeled with N-dansylaziridine, resulting in a fluorescent labeled protein. This derivative (DANZTnI) and native troponin I (TnI) inhibited calmodulin (CaM) stimulation of bovine heart Ca2+-sensitive cyclic nucleotide phosphodiesterase with identical inhibition constants. Association of DANZTnI with CaM was monitored directly by changes in fluorescence intensity in the presence of Ca2+ and by changes in fluorescence anisotropy in the absence of Ca2+. Quantitation of the affinity of CaM for CaM-binding proteins in both the presence and absence of Ca2+ is necessary for prediction of the extent of interaction of both Ca2+ and CaM-binding proteins with CaM in vivo. The Kd for the DANZTnI.cntdot.CaM.cntdot.4Ca2+ and DANZTnI.cntdot.CaM complexes were 20 and 70 .mu.M, respectively. These Kd define a free energy coupling of -4.84 kcal/mol of troponin I for binding of Ca2+ and troponin I to CaM. The Ca2+ dependence for troponin I.cntdot.CaM complex formation predicted from these experimentally determined parameters was closely approximated by the Ca2+ dependence for complex formation between troponin I and fluorescent 5-[[[(iodoacetyl)amino]ethyl]amino]-1-naphthalenesulfonic acid derivatized CaM as determined by fluorescence anisotropy. Complex formation occurred over a relatively narrow range of Ca2+ concentration, indicative of positive heterotropic cooperativity for Ca2+ and troponin I binding to CaM.

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