物理化学学报 >> 2016, Vol. 32 >> Issue (6): 1391-1396.doi: 10.3866/PKU.WHXB201603221

论文 上一篇    下一篇

硫黄素T对淀粉样β-蛋白质40聚集成核动力学的双重影响

李松,刘夫锋,余林玲,赵彦娇,董晓燕*()   

  • 收稿日期:2016-01-04 发布日期:2016-06-03
  • 通讯作者: 董晓燕 E-mail:d_xy@tju.edu.cn
  • 基金资助:
    国家自然科学基金(21236005, 21376172, 21406160, 21576199)

Dual Effect of Thioflavin T on the Nucleation Kinetics of Amyloid β-Protein 40

Song LI,Fu-Feng LIU,Lin-Ling YU,Yan-Jiao ZHAO,Xiao-Yan DONG*()   

  • Received:2016-01-04 Published:2016-06-03
  • Contact: Xiao-Yan DONG E-mail:d_xy@tju.edu.cn
  • Supported by:
    National Natural Science Foundation of China(21236005, 21376172, 21406160, 21576199)

RichHTML

44

PDF

1539

赞

摘要：

荧光染料硫黄素T常用于淀粉样纤维聚集过程的定性定量检测。虽然有研究表明，某些抑制淀粉样蛋白质聚集的小分子抑制剂会与硫黄素T相互作用，影响其测试结果。但硫黄素T如何影响淀粉样蛋白质的聚集成核动力学尚不清晰。本文以淀粉样β-蛋白质40 (Aβ40)为模型，系统研究了硫黄素T对Aβ40聚集成核的影响。研究发现：硫黄素T能够显著改变Aβ40的聚集成核动力学，且影响程度与硫黄素T的浓度密切相关。即在低浓度硫黄素T存在下，Aβ40成核速率的延迟时间先随着硫黄素T浓度的升高而缩短，后随着硫黄素T浓度的升高延迟时间反而延长。但延伸的速率却随硫黄素T浓度的升高而缓慢增大。另外，硫黄素T基本不会影响Aβ40的二级结构和纤维形态。同时，等温滴定微量热实验结果表明，硫黄素T结合Aβ40之间的主要作用力为疏水相互作用。据此，本研究提出硫黄素T对Aβ40聚集成核动力学的双重影响机理。这些结果有助于进一步了解硫黄素T与淀粉样蛋白质的作用特点，为今后硫黄素T在Aβ40聚集成核动力学实验中的使用提供参考。

关键词: 淀粉样β蛋白质, 硫黄素T, 荧光动力学分析, 成核, 分子相互作用

Abstract:

The fluorescent dye thioflavin T (ThT) is widely used for the qualitative and quantitative detection of amyloid fibrils. However, many small-molecular inhibitors have been shown to compete with ThT in binding the fibrils and therefore greatly affect the ThT fluorescence. The effect of ThT on the aggregation kinetics of amyloid proteins is not yet fully understood. Here, using amyloid β-protein 40 (Aβ40) as a model system, we show that ThT significantly alters the aggregation kinetics of Aβ40 in a dose-dependent manner, leading to a decrease-increase trend in the lag time that represents the nucleation rate. Specifically, the lag time decreases as a function of ThT concentration at low ranges, but then begins to increase beyond a specific ThT concentration, which itself increases with Aβ40 concentration. By contrast, the elongation rate slowly increases with ThT concentration. As for the secondary structure and morphology of the fibrils, no significant effects of ThT are observed. Isothermal titration calorimetry suggests that the hydrophobic interaction dominates the binding of ThT to Aβ40. Based on these findings, a working mechanism of the dual effects of ThT on Aβ fibrillization is proposed. These results should aid our understanding of the molecular mechanism of ThT binding with Aβ and allow practical improvements in the measurement of the nucleation kinetics of Aβ fibrillization.

Key words: Amyloid β-protein, Thioflavin T, Fluorescence kinetic assay, Nucleation, Molecular interaction

MSC2000: 

  • O641