Structure, Function and Applications of Inteins

描述(由申请人提供)：将开展一项研究计划，研究内含子如何催化和调节蛋白质剪接和蛋白质反式剪接的各个步骤。蛋白质剪接是一个翻译后的过程，在这个过程中，一个被称为内含子的中间序列被从宿主蛋白质外显子上移除。在蛋白质的反式剪接中，内含子被分成两个片段，只有在这些片段重组时才会发生剪接。内含子存在于所有3个系统发育域的单细胞生物体中，包括几种病原体。此外，所有多细胞生物都含有在成熟过程中发生自蛋白分解反应的蛋白质，这些蛋白质很可能以类似于内含子的方式催化多肽键的分子内断裂。虽然我们对蛋白质剪接中的基本化学步骤有了合理的了解，但我们对内含子如何催化和调节这些步骤的了解还不够充分。因此，有必要研究这一过程的详细机制。这些信息不仅将加深我们对蛋白质剪接及其相关过程的理解，而且对于设计剪接抑制剂和进一步开发蛋白质剪接的实际应用也是至关重要的。在该计划的第一部分，我们建议测试一系列假说(基于上一个资金周期所做的工作)，这些假说与内含子如何协调它们催化的一系列化学步骤以及反式剪接内含子如何高效地相互作用和折叠有关。因此，我们将制备一些含有非天然氨基酸、同位素探针和异肽键的内含素类似物，然后将其用于顺式(目标1)和反式(目标2)蛋白质剪接的动力学、热力学和结构研究。在目标3中，我们将使用定向蛋白质进化方法来分离具有更高活性和更广泛剪接特异性的新的反式剪接内含子。通过作为蛋白质连接酶，这些进化的蛋白质很可能在蛋白质工程中具有广泛的用途。然而，我们生成这些工具的主要动机是提供一种方法来生成包含定义的同位素标记模式的完整膜蛋白，即分段标记，用于核磁共振研究。我们最初的目标将是K+通道KCSA(我们已经研究了几年)，分段标记将用于探索门控机制的各个方面。最终，我们计划将这项技术扩展到其他类别的K+通道和膜蛋白。。与公共卫生相关的蛋白质剪接是包括结核分枝杆菌在内的几种重要人类病原体的基本DNA复制和重组酶成熟所必需的。此外，与蛋白质剪接密切相关的自动加工过程对于所有动物中参与胚胎发育和正常组织动态平衡的蛋白质的脂质修饰是必不可少的，这些蛋白质中的异常活性与人类的各种疾病有关。拟议的蛋白质剪接机制研究将为最终开发这些生物医学相关过程的抑制剂或调节剂以及更快地开发新的生物技术工具奠定基础。

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.