The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid
The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid
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DOI：
10.1021/bi952501g
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发表时间：
1996-05-21
期刊：
影响因子：
2.9
通讯作者：
Kelly, JW
中科院分区：
文献类型：
--
作者：
Lai, ZH;Colon, W;Kelly, JW
甲状腺素运载蛋白（TTR）淀粉样原纤维的形成过程中观察到的部分酸变性，而复性酸变性TTR，这意味着淀粉样原纤维的形成结果从构象中间体的自组装。TTR的酸变性途径已在本文中详细研究，采用多种生物物理方法来表征能够形成淀粉样蛋白原纤维的中间体。在生理浓度下，四聚TTR在pH 7至pH 5之间保持缔合，并且不能形成淀粉样蛋白原纤维。四聚体TTR在依赖于pH和低于pH 5的蛋白质浓度的过程中解离成单体。淀粉样蛋白原纤维形成的程度与TTR单体的浓度相关，TTR单体在5.0-3.9的pH范围内具有改变的但确定的三级结构。TTR的固有Trp荧光监测变性曲线在观察到淀粉样蛋白原纤维形成的pH范围内表现出平台（尽管浓度较高），这意味着检测到具有改变的三级结构的淀粉样蛋白生成中间体的稳态浓度。有趣的是，1-苯胺基-8-萘磺酸盐荧光在与最大淀粉样蛋白原纤维形成相关的pH（pH 4.4）下最小，暗示淀粉样蛋白生成中间体不具有高程度的暴露的疏水表面积，与确定的三级结构一致。甲状腺素运载蛋白在其一级结构中具有两个Trp残基，Trp-41和Trp-79，其方便地位于TTR的三级结构中相距很远。用Phe替换每个Trp提供两个含有单一Trp的变体，其用于探测接近这些发色团的局部pH依赖性三级结构变化。Trp-79-Phe突变体的pH依赖性荧光行为强烈表明Trp-41位于在单体淀粉样蛋白生成中间体形成中发生的三级结构重排的位点附近，可能涉及C链-环-D链区域。在TTR进一步酸化（低于pH 4.4）时，结构确定的单体淀粉样蛋白生成中间体开始采用非淀粉样蛋白生成的替代构象，最终形成低于pH 3的也非淀粉样蛋白生成的A-状态构象。总之，分析平衡超离心、SDS-PAGE、远紫外和近紫外CD、荧光和光散射研究表明，淀粉样蛋白生成中间体是一种具有明确三级结构的单体主要β-折叠结构。
Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate. The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation. At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation. Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5. The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0-3.9. The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration) implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected. Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), impyling that the amyloidogenic intermediate does hot have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure. Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR. Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH-dependent tertiary structural changes proximal to these chromophores. The pH-dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand-loop-D-strand region. Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic. In summary, analytical equilibrium ultracentrifugation, SDS-PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly Beta-sheet structure having a well-defined tertiary structure.