Testing novel hypotheses in Mtu RecA intein splicing

测试 Mtu RecA 内含肽剪接的新假设

基本信息

批准号：
7911738
负责人：
Chunyu Wang
金额：
$ 29.42万
依托单位：
RENSSELAER POLYTECHNIC INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7911738
关键词：
Acids Amides Aspartate Biochemical Biomedical Research Biotechnology C-terminal Catalysis Cells Charge Chemicals Cleaved cell Collaborations Complement component C1s Cysteine Data Dependence Disease Electrostatics Employee Strikes Enzymes Exteins Generations Genetic Goals Histidine Human Hydrogen Bonding Knowledge Ligation Measurement Mediating Methods Mutagenesis Mycobacterium tuberculosis Organism Play Positioning Attribute Process Protein Engineering Protein Precursors Protein Splicing Protons RNA Splicing Reaction Research Research Personnel Role Rotation Solutions Structure Sulfhydryl Compounds Temperature Testing Titrations Toxic effect Vertebral column antimicrobial drug base carboxyl group deprotonation in vivo inhibitor/antagonist intein interdisciplinary approach molecular dynamics mutant novel oxidation programs protonation research study simulation structural biology tool

项目摘要

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.

描述（由申请人提供）：蛋白质剪接是一种精确的翻译后过程，其中中间的蛋白质序列Intein从前体蛋白质中取出，并与侧翼序列N-和C- exteins的结扎伴随结扎。尽管蛋白质剪接的基本步骤是众所周知的，但内部剪接的催化机制仍然很少了解。促进我们对蛋白质剪接的基本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发现了广泛的应用，因此是生物医学研究的必不可少的工具，并有可能用于疾病的治疗。因为只有单细胞生物对于其存活至关重要，因此内素抑制剂可以发展为一种新的抗菌药物，对人类细胞的毒性很少，尤其是对结核分枝杆菌（MTU）。我们提出了MTU RECA Intein中保守残基H73和D422的两个新机械假设。这两个假设得到了来自遗传学，体内剪接数据，溶液NMR研究和计算的各种实验证据的支持。我们的具体目的是结合NMR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检验这两个假设，以证明或反驳这两个新的假设。在此过程中，将生成用于蛋白质剪接的新方法和概念。我们研究的结果将极大地增强我们的理解蛋白质剪接机制，并有助于内部在生物技术中的应用，并有可能在治疗疾病中。长期目标是通过在跨学科方法中应用溶液NMR来研究酶催化，结构，动力学和功能，从而描绘蛋白质剪接的完整催化机制。