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

基本信息

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
项目类别：
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

项目摘要

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.

拟议的工作是我们先前资助的NIH赠款提案的续签。我们的研究重点是酶动力学与催化之间的相互作用。在过去的十二年中，我们开发了技术和探索酶学的一些紧迫问题的方案 - 动力学在催化中的确切作用，跨物种功能动力学的演变以及分子拥挤对催化的影响。我们是使用丙基-TRNA合成酶（pror）作为我们的模型酶，该酶属于称为酶的超家族氨基酰基-TRNA合酶（AARSS）。由于它们在蛋白质合成中的核心作用，AARS出现了作为抗感染药物开发的有吸引力的靶标。生命的所有王国共同，是prors的活跃场所来自不同物种具有显着的序列相似性。结果，针对致病性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，我们是试图利用蛋白质的内在动力学来设计和筛选规格特异性抑制剂病原体。目前，这项工作正处于关头，其中使用最先进的计算进行了彻底的调查技术，标准化的生化方案和良好的光谱方法可以提供对上述主题有更深入的见解。在此，我们提出了一项综合研究，该研究融合了经典，现代的生化和生物物理技术，以解决这些酶学的核心问题。拟议的研究将详细研究静电和动态的相互作用，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。这将通过遵循分子来实现酶的构象动态，底物识别和催化的细节环境。拟议工作的完成可能会导致蛋白质设计和药物的新可能性发现。除了科学相关性外，拟议的工作还将提供出色的机会为我们的学生；参与将产生动手实验室技能的发展，同时加深他们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会导致劳动力的准备，以及在研究生中成长的机会和专业学校。