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: 10797882
• 负责人: Sudeep Bhattacharyay
• 金额: $ 9.99万
• 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/10797882
• 关键词: Affect; Amino Acid Sequence; Amino Acyl-tRNA Synthetases; Anti-Infective Agents; Architecture; Atomic Force Microscopy; Binding; Biochemistry; Biological Assay; Calorimetry; Catalysis; Cells; Chemicals; Coupling; Crowding; Data; Distant; Docking; Drug Design; Drug Targeting; Educational process of instructing; Electrostatics; Environment; Enzymatic Biochemistry; Enzyme Interaction; Enzyme Kinetics; Enzymes; Equilibrium; Equipment; Escherichia coli; Event; Family; Fluorescence Spectroscopy; Grain; In Vitro; Industrialization; Kinetics; Knowledge; Label; Laboratories; Ligand Binding; Measurement; Mediating; Methods; Modeling; Molecular; Molecular Conformation; NMR Spectroscopy; Nature; Nuclear Magnetic Resonance; Organism; Pathogenicity; Pharmaceutical Preparations; Pharmacologic Substance; Physiological; Play; Polymers; Process; Property; Protein Biosynthesis; Protein Conformation; Protein Dynamics; Protein Engineering; Proteins; Quantum Mechanics; Radiolabeled; Reaction; Regulation; Research; Research Project Grants; Role; Sampling; Shapes; Site; Site-Directed Mutagenesis; Solvents; Structure; Students; System; Tertiary Protein Structure; Thermodynamics; Titrations; Uncertainty; Variant; Work; absorption; antimicrobial drug; biophysical chemistry; design; drug development; drug discovery; enzyme model; enzyme structure; experience; experimental study; flexibility; inhibitor; member; molecular dynamics; molecular mechanics; molecular recognition; novel; pathogen; proline-tRNA; quantum; screening; side effect; simulation; small molecule

Broadly speaking, our research group studies the interplay between enzyme dynamics and catalysis. This supplemental proposal is developed to acquire Isothermal Titration Calorimeter, which will aid in investigating the effects of molecular crowding on enzyme structure-dynamics- functions. The interior of a cell is extremely crowded, which can potentially impact protein properties such as structure, folding, stability, ligand binding, and enzyme catalysis. The crowding effects could mediate through hard/steric/entropic and/or soft/chemical/enthalpic interactions. Our objective is to investigate the exact mechanism of crowding/confinement and their impacts on substrate binding and catalysis. We are studying the multidomain prolyl-tRNA synthetases (ProRSs), which play a vital role in protein synthesis in all living organisms. Earlier, we demonstrated that domain dynamics is central to ProRSs functions, which are impacted by the crowder-induced shift in the conformational ensemble. Currently, the impact of the “uniform crowding environment” (nonprotein-based crowders) and “structured crowding environment” (protein-based crowders) on conformational dynamics and function of Escherichia coli (Ec) ProRS is being probed. The mechanistic implications of crowding and confinement effects are significant as these enzymes are promising anti-microbial drug targets. The conformational change and crowder-enzyme interactions are being probed by fluorescence spectroscopy, molecular dynamics simulations, and small molecule docking studies, while varying the size, shape, chemical nature, and concentration of the crowders. We are examining the role of molecular crowders on the substrate binding using Saturation Transfer Difference - Nuclear Magnetic Resonance, which requires a large quantity of proteins and kinetics parameters are obtained from enzyme kinetics assays using radiolabeled substrates. The isothermal titration calorimetry (ITC) method is a label-free direct measurement of the heat evolved or absorbed during a binding event between interacting molecules and enzyme-catalyzed reactions and requires less sample. ITC experiments provide the thermodynamic profiles, which would enable us to establish the molecular mechanism of crowding and confinement resulting in enzyme's altered function. A detailed understanding of crowding effects on the properties of multidomain proteins like Ec ProRS could open up new possibilities for protein designing and drug discovery. Moreover, the ITC equipment would enable us to provide hands-on experiences to students in biochemistry and biophysical chemistry courses (~25 students/semester) by designing course-embedded research projects on calorimetry and teaching them the ligand binding thermodynamics in drug discovery.

概括地说，我们的研究小组研究了酶动力学和 催化作用。本补充方案是为了获得等温滴定量热仪而开发的， 这将有助于研究分子拥挤对酶结构-动力学的影响- 功能。细胞内部极其拥挤，这可能会影响蛋白质 性质，如结构、折叠、稳定性、配体结合和酶催化。拥挤的人群 影响可以通过硬/立体/熵和/或软/化学/焓相互作用来调节。我们的 目的是研究拥挤/限制的确切机制及其对 底物结合和催化。我们正在研究多域的prolyl-tRNA合成酶 (ProRSs)，它在所有生物的蛋白质合成中起着至关重要的作用。此前，我们 演示了结构域动态是PRRSs功能的中心，该功能受到 克劳德诱导的构象系综位移。目前，《军装》的影响 “拥挤环境”(非蛋白质人群)和“结构化拥挤环境” (蛋白质基团)对大肠杆菌(EC)ProRS的构象动力学和功能的影响 正在接受调查。拥挤和限制效应的机制含义是显著的。 因为这些酶是很有希望的抗微生物药物靶点。构象变化和 克劳德-酶的相互作用正在通过荧光光谱进行探测，分子 动力学模拟和小分子对接研究，同时改变大小，形状， 化学性质，以及人群的集中度。我们正在研究分子的作用 饱和转移差-核磁法在衬底结合上的应用 共振需要大量的蛋白质和动力学参数，从 使用放射性标记底物的酶动力学分析。等温滴定量热法(ITC) 方法是对结合事件期间放出或吸收的热量的无标签直接测量 在相互作用的分子和酶催化的反应之间，需要更少的样品。国际贸易中心 实验提供了热力学曲线，这将使我们能够建立 拥挤和限制导致酶功能改变的分子机制。一个 详细了解拥挤效应对EC等多结构域蛋白质性质的影响 ProRS可能为蛋白质设计和药物发现开辟新的可能性。此外， ITC设备将使我们能够为学生提供生物化学和 生物物理化学课程(约25人/学期)的设计课程--嵌入式研究 量热学项目，并教授他们药物发现中的配体结合热力学。

项目成果

Role of Oxidative Stress on SARS-CoV (SARS) and SARS-CoV-2 (COVID-19) Infection: A Review.

• DOI: 10.1007/s10930-020-09935-8
• 发表时间: 2020-12
• 期刊: The protein journal
• 作者: Suhail S;Zajac J;Fossum C;Lowater H;McCracken C;Severson N;Laatsch B;Narkiewicz-Jodko A;Johnson B;Liebau J;Bhattacharyya S;Hati S
• 通讯作者: Hati S

Editing Domain Motions Preorganize the Synthetic Active Site of Prolyl-tRNA Synthetase.

• DOI: 10.1021/acscatal.0c02381
• 发表时间: 2020-09-04
• 期刊: ACS catalysis
• 影响因子: 12.9
• 作者: Hu QH;Williams MT;Shulgina I;Fossum CJ;Weeks KM;Adams LM;Reinhardt CR;Musier-Forsyth K;Hati S;Bhattacharyya S
• 通讯作者: Bhattacharyya S

Cyclic Changes in Active Site Polarization and Dynamics Drive the 'Ping-pong' Kinetics in NRH:Quinone Oxidoreductase 2: An Insight from QM/MM Simulations.

• DOI: 10.1021/acscatal.8b04193
• 发表时间: 2018-11
• 期刊: ACS catalysis
• 影响因子: 12.9
• 作者: Clorice R. Reinhardt;Quin H. Hu;Caitlin G. Bresnahan;S. Hati;S. Bhattacharyya

Evolution of Stronger SARS-CoV-2 Variants as Revealed Through the Lens of Molecular Dynamics Simulations.

• DOI: 10.1007/s10930-022-10065-6
• 发表时间: 2022-10
• 期刊: PROTEIN JOURNAL
• 影响因子: 3
• 作者: Wozney, Alec J.;Smith, Macey A.;Abdrabbo, Mobeen;Birch, Cole M.;Cicigoi, Kelsey A.;Dolan, Connor C.;Gerzema, Audrey E. L.;Hansen, Abby;Henseler, Ethan J.;LaBerge, Ben;Leavens, Caterra M.;Le, Christine N.;Lindquist, Allison C.;Ludwig, Rikaela K.;O'Reilly, Maggie G.;Reynolds, Jacob H.;Sherman, Brandon A.;Sillman, Hunter W.;Smith, Michael A.;Snortheim, Marissa J.;Svaren, Levi M.;Vanderpas, Emily C.;Voon, Aidan;Wackett, Miles J.;Weiss, Moriah M.;Hati, Sanchita;Bhattacharyya, Sudeep
• 通讯作者: Bhattacharyya, Sudeep

Insights into the Mechanism of Tryptophan Fluorescence Quenching due to Synthetic Crowding Agents: A Combined Experimental and Computational Study.

• DOI: 10.1021/acsomega.3c06006
• 发表时间: 2023-11-28
• 期刊: ACS OMEGA
• 影响因子: 4.1
• 作者: Fossum, Carl J.;Johnson, Benjamin O. V.;Golde, Spencer T.;Kielman, Alexis J.;Finke, Brianna;Smith, Macey A.;Lowater, Harrison R.;Laatsch, Bethany F.;Bhattacharyya, Sudeep;Hati, Sanchita
• 通讯作者: Hati, Sanchita