Structure, Function and Applications of Inteins

项目描述（由申请人提供）：研究蛋白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，一个被称为内蛋白的中间序列从宿主蛋白质（外蛋白）上被移除。在蛋白质反式剪接中，内部蛋白被分成两个片段，剪接仅在这些片段重组时发生。蛋白存在于所有3个系统发育域的单细胞生物中，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进行自身蛋白水解反应的蛋白质，这些蛋白质可能以类似于蛋白质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了一个合理的了解，但我们对蛋白质如何催化和调节这些步骤的了解还不太完善。因此，有必要研究这一过程的详细机理。这一信息不仅将加深我们对蛋白质剪接及其相关过程的理解，而且对于剪接抑制剂的设计和蛋白质剪接实际应用的进一步发展至关重要。在项目的第一部分，我们提议测试一系列假设（基于上一个资助周期所做的工作而制定的），这些假设与内部蛋白如何协调它们催化的一系列化学步骤以及反式剪接内部蛋白如何高效地相互作用和折叠有关。因此，我们将制备几种含有非天然氨基酸、同位素探针和异肽键的内部蛋白类似物，然后将它们用于顺式（目的1）和反式（目的2）中蛋白质剪接的动力学、热力学和结构研究。在Aim 3中，我们将采用定向蛋白质进化方法分离具有改进活性和拓宽剪接特异性的新反式剪接蛋白。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，我们生成这些工具的主要动机是提供一种方法来生成包含同位素标记的定义模式的完整膜蛋白，即用于核磁共振研究的片段标记。我们最初的目标将是K+通道KcsA（我们已经研究了几年），片段标记将用于探测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。在包括结核分枝杆菌在内的几种重要的人类病原体中，必需的DNA复制和重组酶的成熟需要蛋白质剪接(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参与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，这些蛋白质的异常活性与人类的各种疾病有关(2)。提出的蛋白质剪接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的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.