Nucleation-dependent aggregation kinetics of Yeast Sup 35 fragment GNNQQNY
Nucleation-dependent aggregation kinetics of Yeast Sup 35 fragment GNNQQNY
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酵母 Sup 35 片段 GNNQQNY 的成核依赖性聚集动力学
DOI：
10.1101/2020.07.27.221150
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Burra G
中科院分区：
文献类型：
--
作者：
Burra G
酵母蛋白Sup35的N端七肽序列GNNQQNY被广泛用作淀粉样原纤维形成的模型系统。在这项研究中，我们使用了一种可重复使用的增溶协议，该协议允许生成GNNQQNY的均一单体溶液，以揭示其自组装机制的分子细节。聚集动力学数据表明，GNNQQNY序列符合成核依赖的聚集动力学，临界核大小为7，而成核效率与反应温度成反比。细胞核通过作为进一步自组装的模板来降低热力学能量障碍，并导致高度有序的淀粉样纤维。在不同温度下生长的纤维表现出类似的硫代黄素T荧光、刚果红结合和富含β片层的结构，表现出特征的交叉β衍射图案。这些聚集体在形态和结构上也与先前报道的相同。成熟的GNNQQNY纤维在与分化的SHSY5Y细胞孵育时没有表现出明显的氧化应激或细胞毒性。据我们所知，这是第一次对基于理论和分子动力学模拟的核大小预测进行实验验证。这些发现为了解与许多系统性和神经退行性疾病相关的淀粉样蛋白/多肽的淀粉样蛋白成核和延长的动力学和热力学提供了基础。
An N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY is widely used as a model system for amyloid fibril formation. In this study, we used a reproducible solubilisation protocol that allows the generation of a homogenous monomeric solution of GNNQQNY to uncover the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequence follows nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the efficiency of nucleation were found to be inversely related to the reaction temperature. The nucleus reduces the thermodynamic energy barrier by acting as a template for further self-assembly and results in highly ordered amyloid fibrils. The fibers grown at different temperatures showed similar Thioflavin T fluorescence, Congo-red binding and β-sheet rich structures displaying a characteristic cross-β diffraction pattern. These aggregates also share morphological and structural identity with those reported earlier. The mature GNNQQNY fibers did not exert significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous nucleus size predictions based on theoretical and molecular dynamics simulations. These findings provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic proteins/peptides associated with many systemic and neurodegenerative diseases.