Interaction of a fluorescent N-dansylaziridine derivative of troponin I with calmodulin in the absence and presence of calcium.
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 nucleodite phosphodiesterase with identical inhibition constants. Association of DANZTnI with calmodulin was monitored directly by changes in flourescence intensity in the presence of Ca2+ and by changes in fluorescence anisotropy in the absence of Ca2+. Quantitation of the affinity of calmodulin for calmodulin-binding proteins in both the presence and absence of Ca2+ is necessary for prediction of the extent of interaction of both Ca2+ and calmodulin-binding proteins with calmodulin in vivo. The dissociation constants for the DANZTnI-calmodulin-l4Ca2+ and DANZTnI-calmodulin complexes were 20 nM and 70 micrometers, respectively. These dissociation constants define a free energy coupling of-4.84 kcal/mol of troponin I for binding of Ca2+ and troponin I to calmodulin. The Ca2+ dependence for troponin I-calmodulin 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-napthalenesulfonic acid derivatized calmodulin 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 calmodulin.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D001388:Aziridines / Q000494:pharmacology*; D001389:Azirines / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins / Q000378:metabolism; D003619:Dansyl Compounds; D005454:Fluorescence Polarization; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D011817:Rabbits; D013816:Thermodynamics; D014336:Troponin / Q000378:metabolism*; D019210:Troponin I
D001388:Aziridines; D001389:Azirines; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D003619:Dansyl Compounds; D009124:Muscle Proteins; D014336:Troponin; D019210:Troponin I; C017173:dansylaziridine; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D002118:Calcium