Kinetic stabilization of the native state by protein engineering: Implications for inhibition of transthyretin amyloidogenesis
Kinetic stabilization of the native state by protein engineering: Implications for inhibition of transthyretin amyloidogenesis
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DOI：
10.1016/j.jmb.2005.01.050
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发表时间：
2005-04-08
影响因子：
5.6
通讯作者：
Kelly, JW
中科院分区：
文献类型：
--
作者：
Foss, TR;Kelker, MS;Kelly, JW
淀粉样蛋白四聚体转甲状腺素（TTR）必须经过限速解离成部分变性单体才能聚集。TTR含有两个不同的四元界面，其中一个定义了甲状腺素和小分子淀粉样蛋白形成抑制剂的结合位点。四聚体的动力学稳定可以通过淀粉样蛋白形成抑制剂选择性地结合到游离过渡态的天然状态或通过将反式抑制亚基（T119M）引入异四聚体以破坏游离过渡态的稳定来实现。在每种情况下，增加离解激活屏障可以防止四聚体离解。在这里，我们证明了拴住两个四元界面定义甲状腺素结合位点的亚基也显着增加了四聚体解离的障碍，显然是通过解离过渡态的不稳定。栓系构建体（TTR- l -TTR）(2)在结构和功能上与野生型TTR相当。尿素不能变性（TTR- l -TTR）(2)，但经GdnHCl处理变性后仍能保持变性状态，说明（TTR- lttr）(2)是动力学稳定的，而不是热力学稳定的，与相同的野生型TTR和（TTR- l -TTR）(2) GdnHCl变性曲线一致。对含有单个TTR-L-TTR链和两个正常单体亚基的结构体的研究表明，仅改变一个四级结构界面就足以对整个四级结构施加动力学稳定。(c) 2005 Elsevier Ltd版权所有。
The amyloidogenic homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to partially denatured monomers in order to aggregate. TTR contains two distinct quaternary interfaces, one of which defines the binding sites for thyroxine and small-molecule amyloidogenesis inhibitors. Kinetic stabilization of the tetramer can be accomplished either by the binding of amyloidogenesis inhibitors selectively to the native state over the dissociative transition state or by the introduction of trans-suppressor subunits (T119M) into heterotetramers to destabilize the dissociative transition state. In each case, increasing the dissociation activation barrier prevents tetramer dissociation. Herein, we demonstrate that tethering two subunits whose quaternary interface defines the thyroxine binding site also dramatically increases the barrier for tetramer dissociation, apparently by destabilization of the dissociative transition state. The tethered construct (TTR-L-TTR)(2) is structurally and functionally equivalent to wild-type TTR. Urea is unable to denature (TTR-L-TTR)(2), yet it is able to maintain the denatured state once denaturation is achieved by GdnHCl treatment, suggesting that (TTR-LTTR)(2) is kinetically rather than thermodynamically stabilized, consistent with the identical wild-type TTR and (TTR-L-TTR)(2) GdnHCl denaturation curves. Studies focused on a construct containing a single TTR-L-TTR chain and two normal monomer subunits establish that alteration of only one quaternary structural interface is sufficient to impose kinetic stabilization on the entire quaternary structure. (c) 2005 Elsevier Ltd. All rights reserved.