The Structural Basis of Transit Peptide Interaction(s) with the Chloroplast Toc Receptors

叶绿体已经进化成半自主的细胞器，其代谢功能在很大程度上依赖于最初由祖先蓝藻基因组编码的核编码蛋白的输入。在叶绿体中起作用的大多数核编码蛋白都是作为具有N-端延伸的高分子量前体合成的，称为转运肽，它介导了前体对叶绿体的特异和有效的靶向。对拟南芥基因组的分析预测，有3000多个前体是以叶绿体为目标的。定义这数千个转运肽的结构和功能标准还不是很清楚。到目前为止，对数百个转运肽的分析未能揭示出显著的保守一级序列，这表明共同的二级或三级结构可能解释了转运肽的特异性靶向活性。在本研究中，我们将利用定点分子突变、核磁共振、等温滴定量热法和分析性超速离心法直接确定拟南芥中肽-受体相互作用的结构基础。该项目将为叶绿体蛋白的输入提供一个新的机制层面的理解。该项目的发现将为其他转运肽的额外结构/功能分析以及它们与叶绿体转运器的其他组件的相互作用提供一个起点。这项研究的一个应用方面可能是加强对新型叶绿体前体蛋白的靶向性，从而提高许多不同农艺植物和植物产品的实用性和安全性。

Plastids have evolved into semi-autonomous organelles whose metabolic function is heavily dependent upon the import of nuclear-encoded proteins that were initially encoded by ancestral cyanobacterial genomes. The majority of nuclear-encoded proteins that function within the plastid are synthesized as higher molecular weight precursors with an N-terminal extension known as a transit peptide, which mediates specific and efficient targeting of the precursor to plastids. Analysis of the Arabidopsis genome predicts that more than 3000 precursors are plastid-targeted. The structural and functional criteria that define these thousands of transit peptides are not well understood. Analysis of hundreds of transit peptides to date has failed to reveal significant conserved primary sequence, suggesting that a common secondary or tertiary structure may account for the specific targeting activity of transit peptides. In this study, site-directed molecular mutagenesis, NMR, isothermal titration calorimetry, and analytical ultracentrifugation will be used to directly determine the structural basis of peptide-receptor interactions in Arabidopsis. This project will provide a new level of mechanistic understanding of chloroplast protein import. Findings from the project will provide a starting point for additional structure/function analysis of other transit peptides and their interaction with other components of the chloroplast translocation apparatus. An applied aspect of this research may be the enhanced targeting of novel plastid precursor proteins to plastids, thereby improving both the utility and safety of many different agronomic plants and plant products.