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Characterization of the interaction between Arabidopsis Toc159 and Toc33 GTPase domains.

机译：拟南芥Toc159和Toc33 GTPase域之间相互作用的表征。

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摘要

Protein-protein interactions are critical for many biochemical processes and for cellular function. Understanding interactions between proteins and ligands, as well as other proteins are necessary for the modelling of biological systems. Protein import into chloroplasts is one such system where the understanding of interactions between members of the translocation complex is limited. The t&barbelow;ranslocon at the o&barbelow;uter envelope membrane of c&barbelow;hloroplasts (Toc complex) consists of three core components: Toc75, Toc159, and Toc34. Members of the Toc159 and Toc34 gene families in Arabidopsis thaliana have been shown to assemble differentially into structurally and functionally distinct complexes. More specifically, there is evidence that complexes containing at Toc159 and at Toc33 (the prefix "at" identifies the species of origin, Arabidopsis thaliana) import photosynthetic proteins, whereas complexes containing at Toc132/120 and at Toc34 seem to preferentially import non-photosynthetic proteins. The interactions between at Toc159 and at Toc33 or at Toc132 and at Toc34 are mediated by their GTPase domains. The current research used recombinant GTPase domains to investigate the specifics of the interaction between at Toc159G and at Toc33G in more detail using molecular and biophysical approaches. Methods included using blue-native PAGE, fluorescence spectroscopy and circular dichroism spectroscopy to characterize the interaction between at Toc159G and at Toc33G. Objectives were to characterize the interaction between at Toc159G and at Toc33G as well as attempt to manipulate the monomer:dimer equilibrium for individual proteins for the purpose of studying heterodimer formation. A secondary objective was to generate single tryptophan mutants of atToc159G (W973F and WI056F) to more precisely characterize the interaction between proteins. Results indicate that only a small percentage of the total protein exists as heterodimers, making quantification using fluorescence spectroscopy and circular dichroism spectroscopy difficult. Nonetheless, it was demonstrated that heterodimer formation occurs between the G-domains of at Toc159 and at Toc33. The data also suggest that higher order oligomers may form, which may be indicative of multiple interaction domains among proteins as previously suggested by stoichiometric studies on isolated Toc complexes. The effects of chelating agents, such as EDTA and Chelex, produced significant structural changes which also inhibited homo-dimerization indicating the specific need for GTP-nucleotide to maintain a functional conformation. Single tryptophan mutants of atToc159G also produced major structural changes to recombinant proteins highlighting the importance of these residues for overall structure of the protein. iii
机译：蛋白质-蛋白质相互作用对于许多生化过程和细胞功能至关重要。了解蛋白质与配体以及其他蛋白质之间的相互作用对于生物学系统的建模是必需的。蛋白质输入到叶绿体中就是这样一种系统，其中对转运复合物成员之间相互作用的理解受到限制。在叶绿体（Toc复合体）的o和barber包膜处的rasconcon由三个核心成分组成：Toc75，Toc159和Toc34。已显示拟南芥中Toc159和Toc34基因家族的成员差异组装成结构和功能上不同的复合物。更具体地说，有证据表明，含有Toc159和Toc33的复合物（前缀“ at”标识起源的拟南芥）输入光合作用蛋白，而含有Toc132 / 120和Toc34的复合物似乎优先输入非光合作用蛋白。蛋白质。 Toc159和Toc33或Toc132和Toc34之间的相互作用是由其GTPase域介导的。当前的研究使用重组GTP酶结构域，以分子和生物物理方法更详细地研究了Toc159G和Toc33G之间相互作用的细节。方法包括使用蓝本色PAGE，荧光光谱和圆二色性光谱来表征Toc159G和Toc33G之间的相互作用。目的是表征Toc159G与Toc33G之间的相互作用，并试图操纵单个蛋白质的单体：二聚体平衡，以研究异二聚体的形成。第二个目标是产生atToc159G的单个色氨酸突变体（W973F和WI056F），以更精确地表征蛋白质之间的相互作用。结果表明，总蛋白中只有一小部分以异二聚体形式存在，这使得使用荧光光谱和圆二色光谱难以定量。尽管如此，已证明异二聚体形成发生在Toc159和Toc33的G结构域之间。数据还表明，可能形成更高阶的低聚物，这可能表明蛋白质之间存在多个相互作用域，如先前对分离的Toc配合物的化学计量研究所建议的那样。螯合剂如EDTA和Chelex的作用产生了显着的结构变化，该结构变化也抑制了同源二聚化，表明对GTP核苷酸保持功能构象的特殊需要。 atToc159G的单个色氨酸突变体也对重组蛋白产生了重大的结构变化，突显了这些残基对蛋白整体结构的重要性。 iii

著录项

  • 作者

    Farquharson, Wesley A.;

  • 作者单位

    Wilfrid Laurier University (Canada).;

  • 授予单位 Wilfrid Laurier University (Canada).;
  • 学科 Biology Molecular.;Biology Plant Physiology.
  • 学位 M.Sc.
  • 年度 2010
  • 页码 73 p.
  • 总页数 73
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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