Structure, Function and Applications of Inteins

内含子的结构、功能和应用

基本信息

批准号：
8321714
负责人：
Tom Muir
金额：
$ 30万
依托单位：
PRINCETON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-01 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8321714
关键词：
Structure Function Applications Inteins

项目摘要

DESCRIPTION (provided by applicant): A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. In the first part of the program we propose to test a series of hypotheses (formulated based on work performed in the last funding cycle) related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we will prepare several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employ these in kinetic, thermodynamic and structural investigations of protein splicing in cis (Aim 1) and in trans (Aim 2). In the Aim 3, we will employ directed protein evolution approaches to isolate new trans-splicing inteins with improved activity and broadened splicing specificities. By acting as protein ligases, these evolved proteins are likely to be of broad utility in protein engineering. However, our primary motivation for generating these tools is to provide a means to generate integral membrane proteins containing defined patterns of isotopic labels, i.e. segmental labeling, for NMR studies. Our initial target will be the K+ channel KcsA (on which we have worked for several years) and segmental labeling will be used to probe aspects of the gating mechanism. Ultimately, we plan to extent this technology to other classes of K+ channel and membrane protein. . PUBLIC HEALTH RELEVANCE Protein splicing is required for the maturation of essential DNA replication and recombination enzymes in several important human pathogens including Mycobacterium tuberculosis (1). In addition, autoprocessing processes closely related to protein splicing are essential for lipid modification of proteins involved in embryonic development and normal tissue homeostasis in all animals, and abnormal activity in these proteins is associated with a variety of disorders in humans (2). The proposed mechanistic investigation of protein splicing will lay the groundwork for the eventual development of inhibitors or modulators of these biomedically relevant processes, as well as the more immediate development of new biotechnology tools.

描述（由申请人提供）：将进行一项研究计划，以研究Inteins如何催化和调节蛋白质剪接和蛋白质反式剪接的各种步骤。蛋白质剪接是一种翻译后过程，其中将内部序列的中间序列从宿主蛋白（外te）中取出。在蛋白质移植中，内部内部的内部分为两块，仅在重构这些片段后才发生剪接。来自所有3个系统发育结构域（包括几种病原体）的单细胞生物中存在于网状素。此外，所有多细胞生物都包含在成熟过程中经历自蛋白解反应的蛋白质，并且可能以类似于输入的方式催化肽键的分子内裂解。虽然我们对蛋白质剪接的基本化学步骤有合理的了解，但我们对Inteins如何催化和调节这些步骤的了解较少。因此，需要研究该过程的详细机制。这些信息不仅将加深我们对蛋白质剪接和相关过程的理解，而且对于剪接抑制剂的设计以及蛋白质剪接实际应用的进一步开发也至关重要。在该计划的第一部分中，我们建议测试一系列假设（基于上一个融资周期中执行的工作制定）与Inteins如何协调它们催化的化学步骤的级联以及移植内部的Inteins如何相互作用和高效折叠。因此，我们将准备几种含有不自然氨基酸，同位素探针和等肽链接的内素类似物，然后在顺式（AIM 1）和反式（AIM 2）中使用这些蛋白质剪接的动力学，热力学和结构研究（AIM 2）。在AIM 3中，我们将采用定向蛋白的演化方法来隔离活性改善并扩大剪接特异性的新型移媒体内部插入。通过充当蛋白质连接酶，这些进化的蛋白质可能在蛋白质工程中具有广泛的效用。但是，我们生成这些工具的主要动机是提供一种方法，以生成包含同位素标签的定义模式的整体膜蛋白，即分段标记，用于NMR研究。我们的最初目标是K+通道KCSA（我们在其上工作了几年），并且将使用分段标记来探测门控机制的方面。最终，我们计划将这项技术用于其他类别的K+通道和膜蛋白。。在包括结核分枝杆菌（1）在内的几种重要人类病原体中，必需的DNA复制和重组酶的成熟是必需的公共卫生相关性蛋白剪接（1）。此外，与蛋白质剪接密切相关的自动处理过程对于所有动物中参与胚胎发育和正常组织稳态的蛋白质的脂质修饰至关重要，这些蛋白质的异常活性与人类的多种疾病有关（2）。拟议的蛋白质剪接机械研究将为这些生物医学相关过程的抑制剂或调节剂的最终开发以及新的生物技术工具的更直接开发奠定基础。