Single-residue mutations have been made of the hydrophobic Ile or Val residue in position 8 of each of the four calcium-binding loop sequences (sites I-IV) of Drosophila calmodulin. These highly conserved residues are part of the hydrophobic core of either calmodulin domain and are involved in the structural link of two calcium-binding sites via a short antiparallel beta-sheet. In the apo-form, the replacement of Ile (or Val) by Gly causes a significant destabilization, shown by the unfolding of the secondary structure of the domain carrying the mutation. In the presence of calcium, the deficiency in alpha-helical structure at 20 degrees C is restored for the mutants at site I, II, or III but not at site IV, which requires the further binding of a high-affinity target peptide to re-establish the native conformation. The extent of the destabilization is seen in the depression of the melting temperature of individual domains, which can be as large as 80 degrees C in the case of Ca4-CaM(V136G). However, because of low values of the unfolding enthalpy for calmodulin domains, only relatively low values of <2 kcal/mol are implied for DeltaDeltaG, the free energy of destabilization due to mutation. Consistent with this, the secondary structure of any unfolded mutant domain is highly sensitive to solvent composition and is largely refolded in the presence of 12.5% (v/v) aqueous trifluoroethanol. Compared to wild-type calmodulin, the affinities of the mutants for calcium and target peptides from sk-MLCK at 20 degrees C are significantly reduced but the effects are relatively small. These results indicate that the conformation of calmodulin can be dramatically altered by mutation of a single highly conserved residue but that changes in solvent or the binding of a target sequence can readily compensate for this, restoring the wild-type properties. The results also suggest that the integrity of both the apo- and holo-forms of calmodulin is important for the maintenance of its biological function and confirm the importance of conserving the structural function of the residues involved in the beta-sheet interactions.

译文

果蝇钙调节蛋白的四个钙结合环序列(I-IV位)中每个位置8的疏水性Ile或Val残基均已形成单残基突变。这些高度保守的残基是任一钙调蛋白结构域的疏水核心的一部分,并通过短的反平行β-折叠参与两个钙结合位点的结构连接。在脱辅基形式中,Ily(或Val)被Gly取代引起明显的不稳定,这由携带突变的域的二级结构的展开显示。在钙的存在下,位点I,II或III但位点IV处的突变体在20℃时α-螺旋结构的缺陷得以恢复,这需要进一步结合高亲和力的靶肽才能重新结合。 -建立本地构象。不稳定的程度体现在单个域的熔化温度降低,对于Ca4-CaM(V136G)而言,可能高达80摄氏度。但是,由于钙调蛋白结构域的解折叠焓值较低,因此对于DeltaDeltaG仅隐含了相对较低的<2 kcal / mol值,这是由于突变引起的去稳定的自由能。与此一致的是,任何未折叠的突变域的二级结构对溶剂组成高度敏感,并在存在12.5%(v / v)的三氟乙醇水溶液的情况下大大重折叠。与野生型钙调蛋白相比,该突变体在20摄氏度下对sk-MLCK的钙和目标肽的亲和力显着降低,但影响相对较小。这些结果表明,钙调蛋白的构象可以通过单个高度保守残基的突变而显着改变,但是溶剂的改变或靶序列的结合可以很容易地弥补这一点,从而恢复野生型特性。该结果还表明钙调蛋白的载脂蛋白和全脂蛋白形式的完整性对于维持其生物学功能非常重要,并确认了保守参与β-sheet相互作用的残基的结构功能的重要性。

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