Project 7: RT-probed protein interaction study in solution

P7。摘要 该项目旨在特定部位表征HIV-1逆转录酶之间的瞬时相互作用 (RT)和它的结合伙伴通过溶液核磁共振，并研究这种相互作用对 相互作用系统的动力学。RT及其配合物的溶液核磁共振研究具有挑战性，给出了 RT异源二聚体的大小。这就需要开发和应用新的方法方法。 适用于大分子络合物的研究。虽然目前的RT X射线结构提供了 关于RT如何与药物和底物相互作用的重要见解，它们是对 通过结晶捕获的构象状态。这些结构不会通知内部的运动 与其功能密切相关的蛋白质或复合体。在这个项目中，我们将填补现有的 知识差距，并决定在RT中的重要功能动态。具体地说，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；以及III)决定生物学上的 RT的结构或动力学变化对IN和核酸结合的意义。要完成以下任务 这个项目的具体目标，我们将采用多管齐下的方法，包括解决方案核磁共振以及 酶学和病毒学研究。我们将结合PCHPI调查人员的互补专业知识 以及合作者和密切协作，以解决与动力的作用有关的重要问题 因HIV感染而接受RT治疗。

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.