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首页> 外文期刊>Biochemistry >Paramecium calmodulin mutants defective in ion channel regulation can bind calcium and undergo calcium-induced conformational switching
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Paramecium calmodulin mutants defective in ion channel regulation can bind calcium and undergo calcium-induced conformational switching

机译：离子通道调控缺陷的草履虫钙调蛋白突变体可结合钙并经历钙诱导的构象转换

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Calmodulin (CaM) is an essential eukaryotic protein that binds calcium ions cooperatively at four EF-hand binding sites to regulate signal transduction pathways. Interactions between the apo domains of vertebrate CaM reduce the calcium affinities ofsites I and II below their intrinsic values, allowing sequential opening of the two hydrophobic clefts in CaM. Viable domain-specific mutants of Paramecium calmodulin (PCaM) differentially affect ion channels and provide a unique opportunity to dissectthe roles of the two highly homologous half-molecule domains. Calcium binding induced an increase in the level of ordered secondary structure and a decrease in Stokes radius in these mutants; such changes were identical in direction to those of wild typeCaM, but the magnitude depended on the mutation. Calcium titrations monitored by changes in the intrinsic fluorescence of Y 138 in site IV showed that the affinities of sites III and IV of wild type PCaM were (i) higher than those of the same sites in rat CaM,(ii) equivalent to those of the same sites in PCaM mutants altered between sites I and II, and (iii) higher than those of PCaM mutants modified in sited III and IV. Thus, calcium saturation drove all mutants to undergo conformational switching inthe same direction but not to the same extent as wild type PCaM. The disruption of the allosteric mechanism that is manifest as faulty channel regulation may be explained by altered properties of switching among the 14 possible partially saturated species of PCaM rather than by an inability to adopt two end-state conformations or target interactions similar to those of the wild type protein.
机译：钙调蛋白（CaM）是一种必不可少的真核蛋白，可在四个EF手结合位点协同结合钙离子，从而调节信号转导途径。脊椎动物CaM的apo结构域之间的相互作用将位点I和II的钙亲和力降低到其固有值以下，从而允许CaM中两个疏水性裂缝的顺序打开。草履虫钙调蛋白（PCaM）的可行域特定突变体差异影响离子通道，并提供了一个独特的机会来剖析两个高度同源的半分子域的作用。钙结合导致这些突变体中有序二级结构水平的增加和斯托克斯半径的降低;此类变化的方向与野生型CaM的方向相同，但幅度取决于突变。通过IV位点Y 138的固有荧光变化监测的钙滴定表明，野生型PCaM的位点III和IV的亲和力（i）高于大鼠CaM中相同位点的亲和力，（ii）等于PCaM突变体中的相同位点在位点I和II之间发生了变化，并且（iii）高于位点III和IV中修饰的PCaM突变体的位点。因此，钙饱和度驱使所有突变体在与野生型PCaM相同的方向上进行构象转换，但程度不同。变构机制的破坏（表现为通道调节错误）可能是由14种可能的部分饱和PCaM物种之间切换特性的改变引起的，而不是由于无法采用与两种构象相似的两种最终状态构象或靶标相互作用而引起的。野生型蛋白。

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