Probing the role of the F-helix in serpin stability through a single tryptophan substitution
Probing the role of the F-helix in serpin stability through a single tryptophan substitution
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DOI：
10.1021/bi0158932
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发表时间：
2002-04-09
期刊：
影响因子：
2.9
通讯作者：
Bottomley, SP
中科院分区：
文献类型：
--
作者：
Cabrita, LD;Whisstock, JC;Bottomley, SP
蛇类通过形成部分折叠的中间体形成环状聚合物。通过诱变和生物物理分析，我们探讨了F-螺旋的构象稳定性，证明它在中间态几乎完全展开。α(1)-抗胰蛋白酶的F-螺旋上的Tyr(160)取代为丙氨酸导致保守的氢键的丧失，这极大地降低了蛋白质对热和溶剂变性的稳定性，表明Tyr160在分子稳定性中的重要性。在α(1)-抗胰蛋白酶的荧光沉默变体中，Tyr160突变为色氨酸残基，产生完全活性、稳定的丝氨酸。对该变异体平衡去折叠行为的荧光分析表明，F-螺旋在中间构象中被高度破坏。碘化物猝灭实验表明，色氨酸残基在中间态和未折叠状态下暴露的程度相同。综上所述，这些数据表明，F-螺旋在控制α(1)-抗胰蛋白酶解离所涉及的早期构象变化方面发挥了重要作用。讨论了这些数据对AT-抗胰蛋白酶功能和错误折叠的影响。
Serpins form loop-sheet polymers through the formation of a partially folded intermediate. Through mutagenesis and biophysical analysis, we have probed the conformational stability of the F-helix, demonstrating that it is almost completely unfolded in the intermediate state. The replacement Of Tyr(160) on the F-helix of alpha(1)-antitrypsin to alanine results in the loss of a conserved hydrogen bond that dramatically reduces the stability of the protein to both heat and solvent denaturation, indicating the importance of Tyr160 in the stability of the molecule. The mutation of Tyr160 to a tryptophan residue, within a fluorescently silent variant of alpha(1)-antitrypsin, results in a fully active, stable serpin. Fluorescence analysis of the equilibrium unfolding behavior of this variant indicates that the F-helix is highly disrupted in the intermediate conformation. Iodide quenching experiments demonstrate that the tryptophan residue is exposed to a similar extent in both the intermediate and unfolded states. Cumulatively, these data indicate that the F-helix plays an important role in controlling the early conformational changes involved in alpha(1)-antitrypsin unfolding. The implications of these data on both at-antitrypsin function and misfolding are discussed.