兔骨骼肌中钙和肌钙蛋白I与肌钙蛋白C结合的能量学

Energetics of the binding of calcium and troponin I to troponin C from rabbit skeletal muscle.

作者信息

Wang C K, Cheung H C

出版信息

Biophys J. 1985 Nov;48(5):727-39. doi: 10.1016/S0006-3495(85)83831-5.

DOI:10.1016/S0006-3495(85)83831-5

PMID:4074834

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1329398/

Abstract

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.

摘要

我们用荧光标记蛋白测定了10℃和20℃时兔骨骼肌肌钙蛋白I（TNI）与肌钙蛋白C（TNC）之间的相互作用自由能。巯基探针5-碘乙酰氨基曙红（IAE）连接到TNC的半胱氨酸（Cys）-98和TNI的Cys-133上，每种标记蛋白都用另一种未标记蛋白进行滴定。在0.3M KCl、pH 7.5、20℃条件下，标记的TNC（TNC*）与TNI形成复合物的缔合常数为6.67×10⁵M⁻¹。在结合Mg²⁺存在时，结合常数增加到4.58×10⁷M⁻¹，在过量Ca²⁺存在时，缔合常数为5.58×10⁹M⁻¹。用未标记的TNC滴定标记的TNI时，得到了非常相似的缔合常数。还在20℃下测定了Ca²⁺与TNC以及复合物TNI×TNC结合的能量学。这两组结果分别用于确定TNI与Mg²⁺或Ca²⁺结合到TNC的耦合自由能。结果得到的总耦合自由能为-5.4千卡。该自由能似乎平均分配到两种物质中：TNI×TNC（Mg）₂或TNI×TNC（Ca）₂，以及TNI×TNC（Ca）₄。相对于游离Ca²⁺的TNI×TNC复合物，前两种物质各自稳定化-2.6千卡，相对于TNI×TNC（Ca）₂或TNI×TNC（Mg）₂，TNI×TNC（Ca）₄稳定化-2.8千卡。耦合自由能显示产生了TNI和TNC之间形成的协同复合物，其中高亲和力位点最初作为游离Ca²⁺的函数被饱和，产生TNI×TNC（Ca）₄。这种饱和发生在游离Ca²⁺浓度范围10⁻⁷至10⁻⁵M内。Ca²⁺结合到TNC的Ca²⁺特异性位点所诱导的TNI和TNC之间连接的协同增强可能与肌动球蛋白ATP酶的激活有直接关系。讨论了Ca²⁺诱导复合物增强所涉及的力的性质。