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Biophysical characterization and structural elucidation of the spectrin-ankyrin interaction.

机译：血影蛋白-锚蛋白相互作用的生物物理表征和结构解析。

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摘要

Organization and membrane integrity in the metazoan cell is maintained through intracellular tethering of membrane proteins to an extensive, underlying, protein network. The principal component of this network, spectrin, is anchored to membrane proteins through the adaptor protein ankyrin. Despite the roles of these membrane skeletal proteins in maintaining cell integrity and polarity, the atomic-level details of their interaction remained unclear. The work presented details the biochemical, biophysical, and structural characterization of the interaction domains of these proteins both individually and as a complex.;To characterize the atomic basis for this interaction, it was first necessary to identify the minimal spectrin and ankyrin binding domains. Previous work had indicated the ankyrin binding region of beta-spectrin to be in the vicinity of repeat 15. Numerous binding assays eventually revealed that tandem beta-spectrin repeats 14 and 15 were necessary to form a stable complex with ankyrin. A simultaneous investigation into the minimal spectrin binding domain of ankyrin was also performed. In the case of ankyrin, previous studies identified two non-overlapping subdomains of ankyrin as possessing binding activity. From a number of in vitro binding assays, it was determined that ZU5-ANK (spanning residues 911-1069) was responsible for high affinity spectrin binding.;After successful characterization of these domains, structural studies were initiated. Crystal structures of the two binding fragments showed that beta-spectrin repeats 14 and 15 maintained a conventional spectrin fold while displaying a large negatively-charged surface patch. ZU5-ANK, on the other hand, exhibited a newly-discovered protein fold and a considerable patch of positively-charged surface residues. Finally, structure determination of the minimal spectrin-ankyrin complex was achieved. Biophysical characterization of clinical, structure-based, and phenotype-inducing mutations verified the structure and provided insight into the nature of a number of clinical mutations.;By focusing on the structure-function relationship of this interaction, a greater understanding of both normal and diseased-state spectrin-ankyrin interactions was provided. While much remains unknown, it is hoped that further studies will continue to detail the roles of spectrin and ankyrin and ultimately advance our knowledge of related diseases such as cardiac arrhythmias, hereditary spherocytosis, elliptocytosis, and malaria.
机译：后生动物细胞的组织和膜完整性通过将膜蛋白与广泛的基础蛋白网络进行胞内束缚而得以维持。该网络的主要成分血影蛋白通过衔接蛋白锚蛋白锚定在膜蛋白上。尽管这些膜骨架蛋白在维持细胞完整性和极性方面发挥着作用，但它们相互作用的原子级细节仍然不清楚。提出的工作详细描述了这些蛋白质相互作用域的生化，生物物理和结构特征，无论是单独还是复合物。为了表征这种相互作用的原子基础，首先必须确定最小的血影蛋白和锚蛋白结合域。先前的研究表明β-血影蛋白的锚蛋白结合区域在重复序列15的附近。许多结合试验最终表明，串联的β-血影蛋白重复序列​​14和15对于与锚蛋白形成稳定的复合物是必需的。同时进行了锚蛋白的最小血影蛋白结合域的研究。就锚蛋白而言，先前的研究确定了锚蛋白的两个非重叠亚域具有结合活性。从许多体外结合试验中，确定ZU5-ANK（跨越残基911-1069）负责高亲和力血影蛋白结合。在成功鉴定了这些结构域后，开始了结构研究。两个结合片段的晶体结构表明，β-血影蛋白重复序列​​14和15保持常规的血影蛋白折叠，同时显示出较大的带负电荷的表面斑。另一方面，ZU5-ANK表现出新发现的蛋白质折叠和大量带正电荷的表面残基。最后，完成了最小血影蛋白-锚蛋白复合物的结构测定。临床，基于结构和表型诱导突变的生物物理特征验证了结构并提供了对许多临床突变本质的洞察力。通过关注这种相互作用的结构-功能关系，可以更好地了解正常和正常情况。提供了病态血影蛋白-锚蛋白相互作用。尽管仍然有许多未知数，但希望进一步的研究将继续详述血影蛋白和锚蛋白的作用，并最终增进我们对相关疾病的了解，例如心律不齐，遗传性球囊增多症，脂细胞增多症和疟疾。

著录项

  • 作者

    Ipsaro, Jonathan James.;

  • 作者单位

    Northwestern University.;

  • 授予单位 Northwestern University.;
  • 学科 Biophysics General.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 217 p.
  • 总页数 217
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:38:26

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