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Mechanism of tocopherol transfer by human alpha-tocopherol transfer protein (alpha-hTTP).

机译:人类α-生育酚转移蛋白(α-hTTP)生育酚转移的机制。

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摘要

Vitamin E is a well known fat soluble chain breaking antioxidant. It is a general term used to describe a family of eight stereoisomers of tocopherols. Selective retention of a-tocopherol in the human circulation system is regulated by the alpha-Tocopherol Transfer Protein (alpha-TTP).;In order to completely avoid OmpF contamination, a GST-alpha-hTTP fusion protein was purified from a glutathione agarose column followed by an on-column thrombin digestion to remove the GST tag. We then demonstrated that alpha-hTTP utilizes a collisional mechanism to deliver its ligand. Furthermore, a higher rate of alpha-tocopherol transfer to small unilamellar vesicles (SUVs) versus large unilamellar vesicles (LUVs) indicated that transfer is sensitive to membrane curvature. These findings suggest that alpha-hTTP mediated alpha-Toc transfer is dominated by the hydrophobic nature of alpha-hTTP and the packing density of phospholipid head groups within acceptor membranes.;Based on the calculated free energy change (AG) when a protein is transferred from water to the lipid bilayer, a model was generated to predict the orientation of alpha-hTTP when it interacts with lipid membranes. Guided by this model, several hydrophobic residues expected to penetrate deeply into the bilayer hydrophobic core, were mutated to either aspartate or alanine. Utilizing dual polarization interferometry and size exclusion vesicle binding assays, we identified the key residues for membrane binding to be F165, F169 and I202. In addition, the rates of ligand transfer of the alpha-TTP mutants were directly correlated to their membrane binding capabilities, indicating that membrane binding was likely the rate limiting step in alpha-TTP mediated transfer of alpha-Toc. The propensity of alpha-TTP for highly curved membrane provides a connection to its colocalization with alpha-Toc in late endosomes.;Using a fluorescently labelled alpha-tocopherol (NBD-alpha-Toc) synthesized in our laboratory, a fluorescence resonance energy transfer (FRET) assay was developed to monitor the kinetics of ligand transfer by alpha-hTTP in lipid vesicles. Preliminary results implied that NBD-alpha-Toc simply diffused from 6-His-alpha-hTTP to acceptor membranes since the kinetics of transfer were not responsive to a variety of conditions tested. After a series of trouble shooting experiments, we identified a minor contaminant, E coli, outer membrane porin F (OmpF) that co-purified with 6-His-alpha-hTTP from the metal affinity column as the source of the problem.
机译:维生素E是众所周知的脂溶性链断裂抗氧化剂。它是一个通用术语,用于描述生育酚的八个立体异构体家族。 α-生育酚转移蛋白(alpha-TTP)调节α-生育酚在人循环系统中的选择性保留;为了完全避免OmpF污染,从谷胱甘肽琼脂糖柱中纯化了GST-alpha-hTTP融合蛋白然后进行柱上凝血酶消化以去除GST标签。然后,我们证明了alpha-hTTP利用碰撞机制来传递其配体。此外,与大单层囊泡(LUVs)相比,向小单层囊泡(SUVs)的α-生育酚转移率更高,表明转移对膜曲率敏感。这些发现表明,α-hTTP介导的α-Toc转移受α-hTTP的疏水性和受体膜内磷脂头基的堆积密度的支配。;基于蛋白质转移时计算出的自由能变化(AG)从水到脂质双层,生成了一个模型来预测α-hTTP与脂质膜相互作用时的方向。在该模型的指导下,预期将深入渗透到双层疏水核中的几个疏水残基突变为天冬氨酸或丙氨酸。利用双偏振干涉法和尺寸排阻囊泡结合测定法,我们确定了膜结合的关键残基为F165,F169和I202。另外,α-TTP突变体的配体转移速率与其膜结合能力直接相关,表明膜结合可能是α-TTP介导的α-Toc转移中的速率限制步骤。 α-TTP对高度弯曲的膜的倾向与其在晚期内体中与α-Toc的共定位有关。;使用在我们实验室中合成的荧光标记的α-生育酚(NBD-alpha-Toc),荧光共振能量转移(开发了FRET)检测方法来监测脂质囊泡中α-hTTP转移配体的动力学。初步结果表明,NBD-α-Toc仅从6-His-α-hTTP扩散至受体膜,因为转移动力学对多种测试条件均无反应。经过一系列的故障排除实验后,我们确定了一种微量污染物,即大肠杆菌,外膜孔蛋白F(OmpF),该杂质与金属亲和柱中的6-His-alpha-hTTP共纯化,是问题的根源。

著录项

  • 作者

    Zhang, Wen Xiao (Wendy).;

  • 作者单位

    Brock University (Canada).;

  • 授予单位 Brock University (Canada).;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 193 p.
  • 总页数 193
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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