SEQUENTIAL CONFORMATIONAL-CHANGES IN CALMODULIN UPON BINDING OF CALCIUM
SEQUENTIAL CONFORMATIONAL-CHANGES IN CALMODULIN UPON BINDING OF CALCIUM
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DOI：
10.1021/bi00304a013
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
STEIN, EA
中科院分区：
文献类型：
--
作者：
BURGER, D;COX, JA;STEIN, EA
[牛]钙调蛋白结合 Ca2+ 时发生的构象变化被分析为 Ca2+ 离子对蛋白质的饱和程度的函数。通过平衡透析并利用Ca2+选择性电极进行Ca与钙调蛋白结合的测定，产生4个化学计量常数，K1=1.16×。 105 M-1，K2 = 2.65 × 105 M-1，K3 = 8.33 × 104 M-1 和 K4 = 1.91 倍。 104 M-1，通过 Adair 方程计算。 4个位点之间的协同效应很小，统计研究表明所有4个结合位点可能是相同且独立的，内在常数为9.31×。 104 M-1。基于这些数据，通过荧光疏水探针分析279和222 nm处的圆二色性变化以及疏水性的出现；两者均作为游离 [Ca2+] 或 Ca2+ 钙调蛋白平均饱和度的函数进行监测。固有酪氨酸椭圆率变化和疏水性暴露伴随着CaM .cntdot 的形成。可以.gtoreq。 2种。作为游离 Ca2+ 和平均饱和度的函数的远紫外圆二色性变化表明每个 CaM .cntdot。 Can物种对信号有不同程度的贡献：α-螺旋含量的半最大增加发生在第一Ca2+的结合上；结合第三个 Ca2+ 时达到最大值。先前的研究表明，只有CaM .cntdot。 Ca3 和 CaM .cntdot。 Ca4 与目标酶相互作用；目前的工作表明主要结构事件发生在步骤 CaM .cntdot 中。 Ca2.fwdarw。 CaM.cntdot。 Ca3是钙调蛋白的靶标可及表面处的疏水板的暴露，其通过高度两亲性和表面寻找的α-螺旋的形成而稳定。
Conformational changes occurring in [bovine] calmodulin on Ca binding were analyzed as a function of the degree of saturation of the protein by Ca2+ ions. Determination of Ca binding to calmodulin, carried out by equilibrium dialyses and by means of a Ca2+-selective electrode, yields 4 stoichiometric constants, K1 = 1.16 .times. 105 M-1, K2 = 2.65 .times. 105 M-1, K3 = 8.33 .times. 104 M-1 and K4 = 1.91 .times. 104 M-1, when calculated by means of the Adair equation. The cooperative effects between the 4 sites are small, and a statistical study reveals that all 4 binding sites may be identical and independent with an intrinsic constant of 9.31 .times. 104 M-1. Based on these data, circular dichroic changes at 279 and 222 nm and the appearance of hydrophobicity was analyzed by means of a fluorescent hydrophobic probe; both were monitored as a function of free [Ca2+] or of the mean saturation of calmodulin by Ca2+. The intrinsic tyrosine ellipticity change and the hydrophobic exposure are concomitant with the formation of the CaM .cntdot. Can .gtoreq. 2 species. The far-UV circular dichroic change as a function of free Ca2+ and of the mean saturation indicates that each CaM .cntdot. Can species contributes to a different extent to the signal: half-maximal increase of the .alpha.-helix content occurs on binding of the 1st Ca2+; the maximal value is reached on binding of the 3rd Ca2+. Previous studies have shown that only CaM .cntdot. Ca3 and CaM .cntdot. Ca4 interact with target enzymes; the present work indicates that the main structural event occurring in the step CaM .cntdot. Ca2 .fwdarw. CaM .cntdot. Ca3 is the exposure of a hydrophobic plate at the target-accessible surface of calmodulin, which is stabilized by the formation of a highly amphiphatic and surface-seeking .alpha.-helix.