We determined the free energy of interaction between rabbit skeletal troponin I (TNI) and troponin C (TNC) at 10 degrees and 20 degrees C with fluorescently labeled proteins. The sulfhydryl probe 5-iodoacetamidoeosin (IAE) was attached to cysteine (Cys)-98 of TNC and to Cys-133 of TNI, and each of the labeled proteins was titrated with the other unlabeled protein. The association constant for formation of the complex between labeled TNC (TNC*) and TNI was 6.67 X 10(5) M-1 in 0.3 M KCl, and pH 7.5 at 20 degrees C. In the presence of bound Mg2+, the binding constant increased to 4.58 X 10(7) M-1 and in the presence of excess of Ca2+, the association constant was 5.58 X 10(9) M-1. Very similar association constants were obtained when labeled TNI was titrated with unlabeled TNC. The energetics of Ca2+ binding to TNC* and the complex TNI X TNC* were also determined at 20 degrees C. The two sets of results were used to separately determine the coupling free energy for binding TNI and Mg2+, or Ca2+ to TNC. The results yielded a total coupling free energy of -5.4 kcal. This free energy appeared evenly partitioned into the two species: TNI X TNC(Mg)2 or TNI X TNC(Ca)2, and TNI X TNC(Ca)4. The first two species were each stabilized by -2.6 kcal, with respect to the Ca2+ free TNI X TNC complex, and TNI X TNC(Ca)4 was stabilized by -2.8 kcal, respect to TNI X TNC(Ca)2 or TNI X TNC(Mg)2. The coupling free energy was shown to produce cooperatively complexes formed between TNI and TNC in which the high affinity sites were initially saturated as a function of free Ca2+ to yield TNI X TNC(Ca)4. This saturation occurred in the free Ca2+ concentration range 10(-7) to 10(-5) M. The cooperative strengthening of the linkage between TNI and TNC induced by Ca2+ binding to the Ca2+-specific sites of TNC may have a direct relationship to activation of actomyosin ATPase. The nature of the forces involved in the Ca2+-induced strengthening of the complex is discussed.
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机译：我们确定了兔骨骼肌钙蛋白I（TNI）和肌钙蛋白C（TNC）在10摄氏度和20摄氏度与荧光标记蛋白相互作用的自由能。巯基探针5-碘乙酰酰胺基曙红（IAE）连接到TNC的半胱氨酸（Cys）-98和TNI的Cys-133，并用其他未标记的蛋白滴定每个标记的蛋白。在0.3 M KCl中，标记的TNC（TNC *）与TNI之间形成复合物的缔合常数为6.67 X 10（5）M-1，在20摄氏度下的pH为7.5。在结合的Mg2 +存在下，结合常数增加到4.58 X 10（7）M-1，并且在存在过量的Ca2 +的情况下，缔合常数为5.58 X 10（9）M-1。当用未标记的TNC滴定标记的TNI时，可获得非常相似的缔合常数。还在20摄氏度下确定了Ca2 +与TNC *和复合TNI X TNC *结合的能量。两组结果分别用于确定将TNI和Mg2 +或Ca2 +与TNC结合的结合自由能。结果产生了-5.4kcal的总耦合自由能。这种自由能似乎均匀地分为两种：TNI X TNC（Mg）2或TNI X TNC（Ca）2和TNI X TNC（Ca）4。相对于不含Ca2 +的TNI X TNC复合物，前两个物种分别稳定在-2.6 kcal，相对于TNI X TNC（Ca）2或TNI X，TNI X TNC（Ca）4稳定在-2.8 kcal。 TNC（镁）2。偶合自由能显示出可在TNI和TNC之间形成协同配合物，其中高亲和力位点最初是作为游离Ca2 +的函数而饱和的，从而产生TNI X TNC（Ca）4。这种饱和发生在游离Ca2 +浓度范围为10（-7）到10（-5）M的区域。由Ca2 +结合到TNC的Ca2 +特异性位点引起的TNI和TNC之间的连接的协同增强可能与肌动球蛋白ATP酶的激活。讨论了Ca2 +诱导的复合物强化作用力的性质。
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