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Determination of the free-energy couplin ... ns and troponin I to calmodulin
Determination of the free-energy coupling for binding of calcium ions and troponin I to calmodulin.
30
Regulation of a wide variety of biological systems by Ca2+ is now known to be mediated through calmodulin, a Ca2+-binding protein. Calmodulin forms Ca2+-dependent complexes with several proteins, including troponin I. We have determined the free-energy coupling (delta GoCT) for binding of Ca2+ and troponin I to calmodulin by measuring Ca2+ binding to calmodulin and to the 1:1 calmodulin-troponin I complex by equilibrium dialysis. The dissociation constant for the Ca2+4.calmodulin and troponin I complex was also determined directly by monitoring fluorescence intensity changes accompanying complex formation between Ca2+4.CaM and N-dansylaziridine-troponin I, a fluorescent derivative of troponin I. Calmodulin displayed four Ca2+-binding sites of similar affinity with a geometric mean dissociation constant of 14 microM. In the presence of saturating troponin I, the geometric mean dissociation constant for the four Ca2+-binding sites was shifted to 1.7 microM. delta GoCT was therefore -1.25 kcal/mol of Ca2+. Saturation of calmodulin with Ca2+ would therefore be expected to increase its affinity for troponin I about 4500-fold. A dissociation constant of 20 nM was determined for the Ca2+4.calmodulin.N-dansylaziridine-troponin I complex. The dissociation constant of the calmodulin.N-dansylaziridine-troponin I complex in the absence of Ca2+ should therefore be about 90 microM. A prediction of this data is that binding of Ca2+ to calmodulin will show strong positive cooperativity when Ca2+ binding to a substoichiometric number of sites is sufficient to promote calmodulin.troponin I complex formation.
Keller CH, Olwin BB, LaPorte DC, Storm DR
Biochemistry
1982-01-05 00:00
21
1
156-62
Calcium,Calcium-Binding Proteins,Calmodulin,Chromatography, Gel,Magnesium,Magnesium Chloride,Muscle Proteins,Osmolar Concentration,Potassium Chloride,Protein Binding,Spectrometry, Fluorescence,Thermodynamics,Troponin,Troponin I,Calcium-Binding Proteins,Calmodulin,Muscle Proteins,Troponin,Troponin I,Magnesium,Calcium,Potassium Chloride,Magnesium Chloride
Biochemistry
NIAID AI 00310, NHLBI HL 05933, NHLBI HL 23606
0006-2960
860
True
7059575
