Exploring the Dynamics of Prolyl-tRNA Synthetases: Towards Developing a Screening Method for Species-Specific Inhibitors

探索脯氨酰-tRNA 合成酶的动力学：开发物种特异性抑制剂的筛选方法

• 批准号: 10203549
• 负责人: Sudeep Bhattacharyay
• 金额: $ 39.73万
• 依托单位: UNIVERSITY OF WISCONSIN EAU CLAIRE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10203549
• 关键词: Active Sites; Address; Affect; Amino Acid Sequence; Amino Acyl-tRNA Synthetases; Anti-Infective Agents; Applications Grants; Architecture; Atomic Force Microscopy; Binding; Biochemical; Biophysics; Calorimetry; Catalysis; Catalytic Domain; Cells; Chemicals; Computational Technique; Coupling; Crowding; Data; Development; Distant; Drug Design; Electrostatics; Entropy; Environment; Enzymatic Biochemistry; Enzyme Kinetics; Enzymes; Equilibrium; Escherichia coli; Evolution; Family; Fluorescence Spectroscopy; Funding; Grain; Human; In Vitro; Industrialization; Investigation; Kinetics; Knowledge; Laboratories; Lead; Life; Ligand Binding; Mechanics; Mediating; Methods; Modeling; Modernization; Molecular; Molecular Conformation; NMR Spectroscopy; Nature; Pathogenicity; Pharmaceutical Preparations; Pharmacologic Substance; Physiological; Play; Polymers; Process; Property; Protein Biosynthesis; Protein Conformation; Protein Dynamics; Protein Engineering; Proteins; Protocols documentation; Readiness; Regulation; Research; Role; Schools; Shapes; Site; Site-Directed Mutagenesis; Solvents; Standardization; Structure; System; Techniques; Titrations; Toxic effect; Uncertainty; United States National Institutes of Health; Ursidae Family; Variant; Work; base; biophysical techniques; design; drug development; drug discovery; enzyme model; experience; flexibility; inhibitor/antagonist; insight; member; molecular dynamics; molecular recognition; novel; pathogen; proline-tRNA; public health relevance; quantum; screening; side effect; simulation; skills; small molecule; student participation

The proposed work is the renewal of our previously funded NIH grant proposal. Our research is focused on the interplay between enzyme dynamics and catalysis. For the past twelve years, we have developed techniques and protocols to explore some pressing issues of enzymology − the precise role of dynamics in catalysis, the evolution of functional dynamics across species, and the impact of molecular crowding on catalysis. We are using prolyl-tRNA synthetase (ProRS) as our model enzyme, which belongs to the superfamily of enzymes called aminoacyl-tRNA synthetases (AARSs). Because of their central role in protein synthesis, AARSs have emerged as attractive targets for anti-infective drug development. Common to all kingdoms of life, the active site of ProRSs from different species bear significant sequence similarity. As a result, drug molecules targeting a pathogenic enzyme's catalytic site could bind to the human counterpart, thus resulting in cell toxicity. Therefore, we are attempting to utilize proteins' intrinsic dynamics for designing and screening species-specific inhibitors for pathogenic ProRSs. Currently, the work is at a juncture, where a thorough investigation using state-of-the-art computational techniques, standardized biochemical protocols, and well-established spectroscopic methods could provide deeper insights into the above-mentioned topics. Herein, we propose a comprehensive study, which blends classical and modern biochemical and biophysical techniques to address these core questions of enzymology. The proposed study will investigate in detail the interplay of electrostatics and dynamics in a quest for the evolutionary origin of the enzyme's catalytic power. Additionally, the proposed study will strive to determine the effects of the crowded cellular milieu on our model enzyme. This will be accomplished by following the molecular details of enzymes' conformational dynamics, substrate recognition, and catalysis in crowded and confined environments. Completion of the proposed work could lead to new possibilities for protein design and drug discovery. In addition to the scientific relevance, the proposed work would provide an outstanding opportunity for our students; participation will yield the development of hands-on laboratory skills while deepening their understanding of these fundamental biophysical concepts. Furthermore, the experience and skills acquired through participation will lead to preparedness for the workforce, as well as opportunities to thrive in graduate and professional schools.

拟议的工作是更新我们以前资助的NIH拨款提案。我们的研究集中在 酶动力学和催化作用之间的相互作用。在过去的12年里，我们开发了 和协议，探索酶学的一些紧迫问题-动力学在催化中的确切作用， 跨物种的功能动力学的演变，以及分子拥挤对催化的影响。我们 使用脯氨酰-tRNA合成酶（ProRS）作为我们的模型酶，其属于酶超家族，称为 氨酰-tRNA合成酶（AARS）。由于它们在蛋白质合成中的核心作用， 作为抗感染药物开发的有吸引力的靶点。所有生命王国都有，ProRS的活性位点 不同物种的序列具有显著的序列相似性。因此，靶向病原体的药物分子 酶的催化位点可以与人类对应物结合，从而导致细胞毒性。因此我们 试图利用蛋白质的内在动力学来设计和筛选物种特异性抑制剂， 致病性ProRS 目前，这项工作正处于一个关键时刻，使用最先进的计算技术进行彻底的调查。 技术，标准化的生化协议，以及完善的光谱方法可以提供 对上述问题有更深入的了解。在此，我们提出了一个全面的研究，其中融合 经典和现代生物化学和生物物理技术来解决酶学的这些核心问题。 拟议的研究将详细调查静电和动力学的相互作用，以寻求 酶催化能力的进化起源此外，拟议的研究将努力确定 拥挤的细胞环境对我们的模型酶的影响。这将通过以下分子来实现： 详细的酶的构象动力学，底物识别，并在拥挤和限制催化 环境.这项工作的完成可能会为蛋白质设计和药物治疗带来新的可能性。 的发现除了科学相关性之外，拟议的工作将提供一个极好的机会， 为我们的学生;参与将有助于发展实践实验室技能，同时加深他们的 了解这些基本的生物物理概念。此外，获得的经验和技能 通过参与将导致劳动力的准备，以及机会，在研究生茁壮成长 和专业学校。