Project 7: RT-probed protein interaction study in solution

项目 7：溶液中 RT 探测的蛋白质相互作用研究

基本信息

批准号：
9407945
负责人：
RIEKO ISHIMA
金额：
$ 23.01万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9407945
关键词：
Affect Amino Acids Avian Leukosis Virus Binding Binding Sites Biological Biological Assay C-terminal Complex Crystallization Crystallography DBL Oncoprotein DNA DNA-Directed RNA Polymerase Defective Viruses Dimerization Enzymatic Biochemistry Enzymes Evolution Fingers Funding HIV HIV Infections HIV-1 Human T-Cell Leukemia Viruses Hybrids Individual Integrase Investigation Knowledge Label Laboratories Ligands Methodology Molecular Molecular Conformation Motion Mutation Nucleic Acid Binding Nucleic Acids Oligonucleotides Pharmaceutical Preparations Polymerase Positioning Attribute Protein Dynamics Proteins RNA RNA-Directed DNA Polymerase Research Personnel Ribonuclease H Roentgen Rays Role Site Structure System Testing Virion Virus Work aptamer base design dimer insight mutant pleiotropism polymerization polypeptide protein function synergism viral DNA virology

项目摘要

P7. Abstract This project aims to site-specifically characterize transient interactions between HIV-1 reverse transcriptase (RT) and its binding partners by solution NMR and to investigate the effects of such interactions on the dynamics of the interacting system. Solution NMR studies of RT and complexes thereof are challenging, given the size of the RT heterodimer. This necessitates developing and applying new methodological approaches suited for investigations of large molecular complexes. While the current RT X-ray structures provide important insights into how RT interacts with drugs and substrate, they are static snapshots of a subset of conformational states captured through crystallization. These structures do not inform on the motions within the protein or complex, which are critically connected to its functions. In this project, we will fill the existing knowledge gap and determine the functionally important dynamics in RT. Specifically, we will i) identify which domains and residues of RT are involved in the interaction with Integrase (IN); ii) delineate specifically the intermolecular contacts on RT and determine the RT/IN dynamics; and iii) determine the biological significance of structural or dynamics changes in RT upon IN and nucleic acid binding. To accomplish the specific aims of this project, we will employ a multi-pronged approach encompassing solution NMR as well as enzymology and virology studies. We will combine the complementary expertise of the PCHPI investigators and collaborators and work in close synergy to tackle the important questions concerning the role of dynamics in RT for HIV infection.

P7。抽象的该项目旨在对 HIV-1 逆转录酶之间的瞬时相互作用进行位点特异性表征 (RT) 及其结合伙伴通过溶液 NMR 并研究这种相互作用对相互作用系统的动力学。 RT 及其复合物的溶液 NMR 研究具有挑战性，因为 RT 异二聚体的大小。这需要开发和应用新的方法论适用于大分子复合物的研究。虽然当前的 RT X 射线结构提供关于 RT 如何与药物和底物相互作用的重要见解，它们是一部分的静态快照通过结晶捕获的构象状态。这些结构不通报内部动议蛋白质或复合物，与其功能密切相关。在这个项目中，我们将填补现有的知识差距并确定 RT 中功能重要的动态。具体来说，我们将 i) 确定哪些 RT 的结构域和残基参与与整合酶 (IN) 的相互作用； ii) 具体描述 RT 上的分子间接触并确定 RT/IN 动力学； iii) 确定生物学 IN 和核酸结合后 RT 的结构或动力学变化的重要性。为了实现为了实现该项目的具体目标，我们将采用多管齐下的方法，包括溶液核磁共振以及酶学和病毒学研究。我们将结合 PCHPI 研究人员的互补专业知识与合作者密切协同工作，解决有关动态作用的重要问题接受 HIV 感染 RT 治疗。