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

质体已经进化成半自主的细胞器，其代谢功能严重依赖于最初由祖先蓝藻基因组编码的核编码蛋白的进口。大多数在质体内起作用的核编码蛋白是作为高分子量前体合成的，其n端延伸被称为传递肽，它介导了对质体前体的特异性和有效靶向。对拟南芥基因组的分析预测，超过3000个前体是质体靶向的。定义这数千个转运肽的结构和功能标准尚不清楚。迄今为止，对数百个转运肽的分析未能揭示重要的保守一级序列，这表明一个共同的二级或三级结构可能解释了转运肽的特定靶向活性。在本研究中，将使用位点定向分子诱变、核磁共振、等温滴定量热法和分析性超离心来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输入的机制理解提供一个新的水平。该项目的研究结果将为其他转运肽的额外结构/功能分析及其与叶绿体易位装置其他组分的相互作用提供起点。该研究的一个应用方面可能是增强新的质体前体蛋白对质体的靶向性，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.