Phenylalanine fluorescence studies of calcium binding to N-domain fragments of Paramecium calmodulin mutants show increased calcium affinity correlates with increased disorder.
Phenylalanine fluorescence studies of calcium binding to N-domain fragments of Paramecium calmodulin mutants show increased calcium affinity correlates with increased disorder.
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钙与草履虫钙调蛋白突变体 N 结构域片段结合的苯丙氨酸荧光研究表明，钙亲和力的增加与紊乱的增加相关。
DOI：
10.1110/ps.11601
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Shea,MA
中科院分区：
文献类型：
--
作者：
VanScyoc,WS;Shea,MA
钙调蛋白(CaM)是一种普遍存在的必需的钙结合蛋白，它调节不同的蛋白质靶点以响应生理的钙波动。大多数CaM-靶复合体的高分辨结构表明，CaM的两个同源结构域在目标识别中是等价的伙伴。然而，草履虫钙调蛋白(PCaM)N区钙结合位点I和II之间的突变选择性地影响钙依赖的钠电流。为了了解这些结构域特异性的影响，对其中6个突变体的N-结构域片段(PCaM1-75)进行了检测，以确定钙与I和II位点结合的能量学或构象性质是否受到干扰。这些PCaM(1-75)序列自然含有5个Phe残基，但没有Tyr或Trp；钙结合是通过观察280 nm处本征苯丙氨酸荧光的减少来监测的。为了评估突变诱导的构象变化，测定了apo PCaM(1-75)序列的热变性和Stokes半径中钙依赖的变化。各突变体的钙结合自由能均在野生型的1千卡/摩尔范围内，钙降低了所有突变体的Rs值。观察到一个显着的趋势，表现出钙亲和力增加和RS的突变体伴随着热稳定性的下降(高达18°C)。因此，在载脂蛋白状态下增加无序性和减少三级约束的结合位点之间的突变促进了钙的协调。这一发现突显了钙结合和构象变化之间联系的复杂性，以及预测突变效应的难度。
Calmodulin (CaM) is a ubiquitous, essential calcium‐binding protein that regulates diverse protein targets in response to physiological calcium fluctuations. Most high‐resolution structures of CaM‐target complexes indicate that the two homologous domains of CaM are equivalent partners in target recognition. However, mutations between calcium‐binding sites I and II in the N‐domain ofParameciumcalmodulin (PCaM) selectively affect calcium‐dependent sodium currents. To understand these domain‐specific effects, N‐domain fragments (PCaM1–75) of six of these mutants were examined to determine whether energetics of calcium binding to sites I and II or conformational properties had been perturbed. These PCaM(1–75)sequences naturally contain 5 Phe residues but no Tyr or Trp; calcium binding was monitored by observing the reduction in intrinsic phenylalanine fluorescence at 280 nm. To assess mutation‐induced conformational changes, thermal denaturation of the apo PCaM(1–75)sequences, and calcium‐dependent changes in Stokes radii were determined. The free energy of calcium binding to each mutant was within 1 kcal/mole of the value for wild type and calcium reduced the Rsof all of them. A striking trend was observed whereby mutants showing an increase in calcium affinity and Rshad a concomitant decrease in thermal stability (by as much as 18°C). Thus, mutations between the binding sites that increased disorder and reduced tertiary constraints in the apo state promoted calcium coordination. This finding underscores the complexity of the linkage between calcium binding and conformational change and the difficulty in predicting mutational effects.