Testing novel hypotheses in Mtu RecA intein splicing

描述(申请人提供)：蛋白质剪接是一个精确的翻译后过程，在这个过程中，插入的蛋白质序列，内含子，从前体蛋白中移除，伴随着侧翼序列，N-和C-外显子的连接。虽然蛋白质剪接的基本步骤已经众所周知，但内含素剪接的催化机制仍然知之甚少。提高我们对蛋白质剪接的基础知识可以产生两个主要影响：Inteins在蛋白质工程和生物技术中得到了广泛的应用，因此是生物医学研究和潜在的疾病治疗中不可或缺的工具。由于只有单细胞生物才有对其生存至关重要的内含素，因此内含素抑制剂可以发展成为一类对人类细胞毒性很小的新型抗菌药物，特别是对结核分枝杆菌(MTU)。我们对MTU RecA整合素中的保守残基H73和D422提出了两个新的力学假设。这两个假说已经得到了来自遗传学的各种实验证据、体内内含子突变体的剪接数据、溶液核磁共振研究和计算的支持。我们的具体目标是使用核磁共振结构生物学方法、剪接反应的生化表征和分子动力学模拟来验证这两个假说，以证明或驳斥这两个新的假说。在这个过程中，将产生蛋白质剪接的新方法和新概念。我们的研究结果将极大地加深我们对蛋白质剪接机制的理解，并有助于内含子在生物技术中的应用，以及潜在的治疗疾病。长期目标是通过应用溶液核磁共振在研究酶催化、结构、动力学和功能的跨学科方法中描绘蛋白质剪接的完整催化机制。

DESCRIPTION (provided by applicant): Protein splicing is a precise post-translational process in which an intervening protein sequence, intein, is removed from a precursor protein with the concomitant ligation of the flanking sequences, N- and C-exteins. Although the basic steps of protein splicing are well-known, the catalytic mechanisms of intein splicing are still poorly understood. Advancing our fundamental knowledge of protein splicing can have two major impacts: Inteins have found extensive applications in protein engineering and biotechnology and therefore are an indispensable tool for biomedical research and potentially for therapies of diseases. Because only unicellular organisms have inteins vital for their survival, intein inhibitors can develop into a new class of antimicrobial drug with little toxicity for human cells, especially for Mycobacterium tuberculosis (Mtu). We propose two new mechanistic hypotheses for conserved residues H73 and D422 in Mtu RecA intein. These two hypotheses have been supported by diverse experimental evidence from genetics, in vivo splicing data in intein mutants, solution NMR studies and by calculations. Our specific aims are to test these two hypotheses using a combination of NMR structural biology methods, biochemical characterization of splicing reaction and molecular dynamics simulation to prove or refute these two novel hypotheses. In the process, new methods and concepts for protein splicing will be generated. The results from our research will greatly enhance our understanding the mechanisms of protein splicing and contribute to the application of inteins in biotechnology and potentially in treating diseases. The long term goal is to delineate the complete catalytic mechanisms of protein splicing by applying solution NMR in an interdisciplinary approach for studying enzyme catalysis, structure, dynamics and function.