Specificity in Class I Aminoacyl-tRNA Synthetasis

I 类氨酰基-tRNA 合成的特异性

• 批准号: 6361734
• 负责人: JOHN J. PERONA
• 金额: $ 26.33万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6361734
• 关键词: X ray crystallography; active sites; aminoacid tRNA ligase; bacterial proteins; biochemistry; chemical binding; chemical kinetics; conformation; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; molecular biology; molecular shape; protein engineering; protein structure function; site directed mutagenesis; structural biology

DESCRIPTION (provided by applicant): A multidasciplinary study aimed at uncovering the structural and energetic basis for amino acid and transfer RNA specificity in several class I aminoacyl-tRNA synthetases is proposed. The experimental plan will focus on the structure-based design of directed modifications in both the synthetase and tRNA, and on the insightful analysis of modified complexes by a variety of enzymological and biophysical methods. The role of the globular tertiary core region of glutamine-specific tRNA in aminoacylation will be explored by exploiting the availability of an extensive tRNA sequence database, as well as by the introduction of highly specific chemical modifications to the sugar-phosphate backbone. Glutaminylation kinetics, together with high-resolution structures bound to synthetase and to Ef-Tu, will be determined to assess the effects of the alterations. In a separate line of experiments, rational structure-based approaches are planned to re-engineer the amino acid specificity from glutamine to glutamate. New methodologies will be applied to improve the chemical and enzymatic synthesis of multimilligram quantities of homogeneous RNAs, and to better characterize the aminoacylation reaction by implementing a new assay useful for both steady-state and transient kinetic measurements. With improved kinetic methods in hand, a functional analysis of glutaminyl-tRNA synthetase will be pursued with the aim of addressing the kinetic basis for the amino acid specificity, the function of divalent metal ions, and the requirement for tRNA to carry out activation of the amino acid. These approaches will also be used to examine kinetic properties of the homologous cysteinyl-tRNA synthetase. Detailed information on the origins of class I bacterial tRNA synthetase specificities will aid drug-design approaches which exploit the structural differences between bacterial and human synthetases to develop antimicrobial agents. Re-engineering of the amino acid specificities of tRNA synthetases is also the most challenging step in the in viva production of proteins containing non-natural amino acids. Because of the broad applicability of this technology, the development of new synthetases based on the class I active site domain has potential for impact on biomedical research toward therapies for many human diseases.

描述（由申请人提供）：一项多学科研究，旨在 揭示氨基酸和转移RNA的结构和能量基础 提出了几种 I 类氨酰基-tRNA 合成酶的特异性。这 实验计划将重点关注定向结构的设计 合成酶和 tRNA 的修饰以及深入的分析 通过各种酶学和生物物理方法修饰复合物。 谷氨酰胺特异性 tRNA 球状三级核心区在 将通过利用广泛的可用性来探索氨酰化 tRNA 序列数据库，以及通过引入高度特异性 对糖-磷酸骨架进行化学修饰。谷氨酰化 动力学，以及与合成酶结合的高分辨率结构 将确定 Ef-Tu 以评估变更的影响。在一个 单独的实验线，计划基于合理结构的方法 重新设计从谷氨酰胺到谷氨酸的氨基酸特异性。新的 将应用方法来改进化学和酶合成 数毫克数量的均质 RNA，并更好地表征 通过实施对两者都有用的新测定法来进行氨酰化反应 稳态和瞬态动力学测量。通过改进的动力学方法 目前，将对谷氨酰胺酰-tRNA 合成酶进行功能分析 目的是解决氨基酸特异性的动力学基础， 二价金属离子的功能以及tR​​NA执行的要求 氨基酸的活化。这些方法也将用于检查 同源半胱氨酰-tRNA 合成酶的动力学特性。详细的 I 类细菌 tRNA 合成酶特异性起源的信息 将有助于利用结构差异的药物设计方法 细菌和人类合成酶之间的相互作用，以开发抗菌剂。 tRNA合成酶氨基酸特异性的重新设计也是 体内生产含有的蛋白质中最具挑战性的一步 非天然氨基酸。由于这项技术的广泛适用性， 基于 I 类活性位点域的新合成酶的开发 对许多人类治疗的生物医学研究产生影响的潜力 疾病。