Structure, Function and Applications of Inteins

描述（由申请人提供）：将进行一项研究计划，以研究内含肽如何催化和调节蛋白质剪接和蛋白质反式剪接中的各个步骤。蛋白质剪接是一个翻译后过程，其中一个插入序列，称为内含肽，从宿主蛋白质，外显肽去除。在蛋白质反式剪接中，内含肽被分成两部分，并且剪接仅在这些片段重构时发生。内含肽存在于所有3个系统发育域的单细胞生物体中，包括几种病原体。此外，所有多细胞生物体都含有在成熟过程中经历自蛋白水解反应的蛋白质，这些蛋白质可能以类似于内含肽的方式催化分子内肽键的裂解。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，但我们对内含肽如何催化和调节这些步骤的知识还不太发达。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和相关过程的理解，而且对剪接抑制剂的设计和蛋白质剪接实际应用的进一步发展也至关重要。在该计划的第一部分，我们建议测试一系列假设（基于上一个资助周期中进行的工作制定），这些假设与内含肽如何协调它们催化的化学步骤级联以及反式剪接内含肽如何相互作用并高效折叠有关。因此，我们将准备几个内含肽类似物含有非天然氨基酸，同位素探针和异肽连接，然后采用这些在动力学，热力学和结构研究蛋白质剪接顺式（目标1）和反式（目标2）。在目标3中，我们将采用定向蛋白质进化的方法来分离新的反式剪接内含肽，其具有改善的活性和拓宽的剪接特异性。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用途。然而，我们产生这些工具的主要动机是提供一种方法来产生包含同位素标记的定义模式的完整膜蛋白，即片段标记，用于NMR研究。我们最初的目标将是K+通道KcsA（我们已经工作了几年）和节段标记将用于探测方面的门控机制。最终，我们计划将这项技术扩展到其他类型的K+通道和膜蛋白。.公共卫生相关性在包括结核分枝杆菌在内的几种重要的人类病原体中，蛋白质剪接是必需的DNA复制和重组酶成熟所必需的（1）。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涉及胚胎发育和正常组织稳态的蛋白质的脂质修饰是必不可少的，并且这些蛋白质中的异常活性与人类中的各种疾病有关（2）。蛋白质剪接的机制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生物技术工具的更直接开发奠定基础。

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.