Determination of the free-energy coupling for binding of calcium ions and troponin I to calmodulin.
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.
D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002850:Chromatography, Gel; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009124:Muscle Proteins / Q000378:metabolism*; D009994:Osmolar Concentration; D011189:Potassium Chloride; D011485:Protein Binding / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014336:Troponin / Q000378:metabolism*; D019210:Troponin I
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D009124:Muscle Proteins; D014336:Troponin; D019210:Troponin I; D015636:Magnesium Chloride; D011189:Potassium Chloride; D008274:Magnesium; D002118:Calcium