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

拟议的工作是我们之前资助的 NIH 拨款提案的更新。我们的研究重点是 酶动力学和催化之间的相互作用。在过去的十二年里，我们开发了技术 以及探索酶学一些紧迫问题的协议——动力学在催化中的精确作用、 跨物种功能动力学的演化，以及分子拥挤对催化的影响。我们是 使用脯氨酰-tRNA 合成酶 (ProRS) 作为我们的模型酶，它属于酶超家族，称为 氨酰基-tRNA 合成酶 (AARS)。由于它们在蛋白质合成中的核心作用，AARS 已经出现 作为抗感染药物开发的有吸引力的目标。 ProRS 的活性位点是所有生命王国所共有的 来自不同物种的序列具有显着的相似性。因此，针对致病菌的药物分子 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 尝试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，即使用最先进的计算技术进行彻底的调查 技术、标准化生化方案和完善的光谱方法可以提供 对上述主题有更深入的了解。在此，我们提出一项综合研究，其中融合了 经典和现代的生物化学和生物物理技术来解决酶学的这些核心问题。 拟议的研究将详细研究静电学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。这将通过遵循分子 拥挤和有限环境中酶的构象动力学、底物识别和催化的细节 环境。完成拟议的工作可能会为蛋白质设计和药物带来新的可能性 发现。除了科学相关性之外，拟议的工作还将提供一个绝佳的机会 为了我们的学生；参与将促进实验室实践技能的发展，同时加深他们的知识 了解这些基本的生物物理学概念。此外，所获得的经验和技能 通过参与将为劳动力做好准备，并为毕业生提供蓬勃发展的机会 和专业学校。

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.