Autophosphorylation dependent destabilization of the insulin receptor kinase domain: tryptophan-1175 reports changes in the catalytic cleft.
Autophosphorylation dependent destabilization of the insulin receptor kinase domain: tryptophan-1175 reports changes in the catalytic cleft.
复制标题
胰岛素受体激酶结构域的自磷酸化依赖性不稳定：色氨酸-1175 报告催化裂隙的变化。
DOI：
10.1021/bi982546s
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发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Kohanski,RA
中科院分区：
文献类型：
--
作者：
Bishop,SM;Ross,JB;Kohanski,RA
蛋白激酶受构象或化学变化的调节，这些构象或化学变化促进底物接近活性位点并促进催化必需残基和水分子的正确取向。胰岛素受体激酶结构域（IRKD）的基础和激活状态之间的转换是由自身磷酸化引起的。通过盐酸胍（GdnHCl）依赖性变性和碘对内源荧光的猝灭，研究了IRKD自磷酸化对构象稳定性的影响。重组可溶性IRKD的色氨酸残基（残基R953-S1355）在λ ex 295 nm处激发，并分析发射光谱的质心（吲哚环环境的平均极性特征）和在λ em 310 - 420 nm范围内的积分荧光强度。载脂蛋白和磷酸化IRKD形式的变性概况是复杂的，至少有三个不同的展开过渡。第一个和最后一个过渡是可逆的和合作，并在0.4或0.7 M盐酸钆和2.4或2.7 M盐酸钆的中点，分别发生在较低的盐酸钆浓度的磷酸-IRKD的过渡。解折叠自由能的计算表明，第一次跃迁的稳定性损失为1.23kcal/mol，第三次跃迁的稳定性损失为1.5kcal/mol。圆二色性显示在第一个过渡和全球展开的最后一个过渡的二级结构的微妙变化。第一个过渡报告的变化主要是在本地环境的W1175，这是附近的催化环，是保守的蛋白酪氨酸激酶。W1175也是天然发射光谱的主要荧光团。碘淬灭W1175在apo-IRKD中几乎检测不到，但在磷酸化IRKD中显著，表明W1175暴露于小溶质强烈依赖于激活环的构象。这些研究表明，自磷酸化，而暴露的催化中心，也产生了构象稳定性低于脱辅基酶。
Protein kinases are regulated by conformational or chemical changes which facilitate access of substrates to the active site and promote correct orientations of catalytically essential residues and water molecules. The switch between basal and activated states of the insulin receptor's kinase domain (IRKD) results from autophosphorylation. We investigated the effects of IRKD autophosphorylation on the conformational stability by guanidine hydrochloride (GdnHCl) dependent denaturation and by iodide quenching of intrinsic fluorescence. Tryptophan residues of the recombinant soluble IRKD (residues R953−S1355) were excited at a λexof 295 nm, and emission spectra were analyzed for centroid (a characteristic of average polarity of the indole rings' environments) and integrated fluorescence intensity over the λemrange of 310−420 nm. Denaturation profiles of both apo- and phospho-IRKD forms are complex with at least three distinct unfolding transitions. The first and last transitions were reversible and cooperative and had midpoints at 0.4 or 0.7 M GdnHCl and 2.4 or 2.7 M GdnHCl, respectively; transitions of phospho-IRKD occurred at lower GdnHCl concentrations. Calculations of free energy of unfolding suggested a loss of ∼2.3 kcal/mol of stabilization for the first transition and ∼1.5 kcal/mol for the third transition. Circular dichroism showed subtle changes in secondary structure over the first transition and global unfolding over the last transition. The first transition reports changes primarily in the local environment of W1175, which is near the catalytic loop and is conserved among protein tyrosine kinases. W1175is also the dominant fluorophore of the native emission spectrum. Iodide quenching of W1175was virtually undetectable in the apo-IRKD but significant in the phospho-IRKD, suggesting that W1175exposure to small solutes is strongly dependent on the conformation of the activation loop. These studies indicate that autophosphorylation, while exposing the catalytic center, also produces a conformer less stable than the apoenzyme.