HDX and NMR Core

HDX 和 NMR 核心

• 批准号: 10242903
• 负责人: Patrick Robert Griffin
• 金额: $ 104.57万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242903
• 关键词: 3-Dimensional; 5' Untranslated Regions; Achievement; Address; Affect; Affinity; Amino Acid Sequence; Anus; Binding; Binding Sites; Biological Process; C-terminal; Collection; Communication; Complex; Cryoelectron Microscopy; Custom; DBL Oncoprotein; DNA; Data; Databases; Deuterium; Disease; Elderly; Enzyme Inhibition; Evolution; Family; Funding; Goals; HIV; HIV-1; HIV-1 integrase; HIV-1 protease; Human; Hydrogen; Integrase; Integrase Inhibitors; Isotopes; Kinetics; Length; Lentivirus; Ligand Binding; Ligands; Manuscripts; Mass Spectrum Analysis; Molecular Conformation; Molecular Probes; Motion; Nuclear Magnetic Resonance; Nucleic Acids; Peptide Fragments; Polymerase; Post-Translational Protein Processing; Preparation; Process; Property; Protein Conformation; Protein Dynamics; Proteins; RNA; RNA-Directed DNA Polymerase; Reporting; Research Personnel; Roentgen Rays; Role; Sampling; Scientist; Spumavirus; Structure; Techniques; Technology; Therapeutic Intervention; Thiophenes; Transfer RNA; Viral; Virus; X-Ray Crystallography; aptamer; base; design; gag-pol Fusion Proteins; genetic regulatory protein; inhibitor/antagonist; innovation; member; molecular recognition; pol Gene Products; protein complex; protein structure; prototype; quinoline; receptor; resistance mutation; small molecule; tandem mass spectrometry; therapeutic protein; transcriptional coactivator p75; viral RNA

Abstract Proteins are dynamic molecules that undergo constant flexing and motion. Changes in protein conformation and dynamics are important to molecular recognition and these changes occur during many key biological processes including activation and inhibition of enzymes, ligand binding to receptors, posttranslational modifications, and allosteric communication across protein-protein interfaces. All of these processes are regulated through the structure and dynamics of the proteins within the functional complex. Thus, techniques to study protein structure and dynamics are critical to the understanding of biological processes and to aid in developing strategies to target proteins for therapeutic intervention in disease. The HDX-NMR Core will address central goals of the HIVE Center that include characterization of HIV protein interactions among themselves, with host molecules, or with small molecule probes. A key focus of HDX efforts will be the structure and dynamics of the HIV-1 reverse transcriptase (RT) initiation complex, or RTIC, a large protein-nucleic acid complex comprising RT (p66/p51 heterodimer), the primer-binding site region of the 5’ untranslated viral RNA (vRNA) and a human tRNA (Project 3: Arnold, Musier-Forsyth, Griffin, Lyumkis, and Sarafianos). We will also investigate the interactions of HIV-1 RT with members of the APOBEC3 (A3) family of restriction factors (most notably A3G), which have been reported to suppress the polymerase activity of RT and will also be studied in Project 3. In addition to the RTIC from lentivirus HIV-1, we will also investigate the RTIC of spumavirus prototype foamy virus (PFV). We are taking a multifaceted approach towards understanding these important virus-host complex interactions, using cryo-EM, X-ray crystallography, HDX, SAXS, and pre-steady state kinetic analyses. Among the main questions addressable by HDX are: How does the vRNA/tRNA interact with RT compared to DNA/DNA and DNA/RNA substrates (cryo-EM, X-ray, HDX, kinetics)? What is the role of RNA structure in this complex and how does it interact with RT (cryo-EM, X-ray, HDX)? What are the dynamic properties of the initiation complex (cryo-EM, SAXS, HDX, kinetics)? How do interactions in the HIV-1 and PFV RTICs differ? How does HIV-1 RT interact with A3 proteins to contribute to HIV-1 restriction? HDX has been instrumental for studying quinoline-based allosteric integrase (IN) inhibitor (ALLINIs) induced aberrant multimerization of full-length wild type (WT) IN and its application will now be extended to examine how recently discovered (Core 4) thiophene- based inhibitors alter the IN structure (Project 1). HDX-MS will also be used to probe inter-protein interactions among HIV-1 Gag and Gag-Pol proteins (Project 2), the effect of resistance mutations or inter-clade differences in inter-protein interactions in HIV-1 proteins (Project 5), and interactions between HIV-1 proteins and SuFEx or other small molecule probes (Project 6). NMR will be used to support Project 6 and Core 4.

抽象的 蛋白质是动态分子，蛋白质构象不断弯曲和运动。 和动力学对于分子识别很重要，这些变化发生在许多关键的生物过程中 过程包括酶的激活和抑制、配体与受体的结合、翻译后 所有这些过程都是蛋白质-蛋白质界面之间的修饰和变构通讯。 通过功能复合物内蛋白质的结构和动力学进行调节。 研究蛋白质结构和动力学对于理解生物过程和帮助 HDX-NMR 核心将解决开发针对疾病治疗干预的蛋白质策略。 HIVE 中心的中心目标包括 HIV 蛋白之间相互作用的表征， HDX 工作的重点是结构和动力学。 HIV-1 逆转录酶 (RT) 起始复合物或 RTIC（一种大型蛋白质-核酸复合物）的组成 包含 RT（p66/p51 异二聚体）、5’非翻译病毒 RNA (vRNA) 的引物结合位点区域和 我们还将研究人类 tRNA（项目 3：Arnold、Musier-Forsyth、Griffin、Lyumkis 和 Sarafianos）。 HIV-1 RT 与 APOBEC3 (A3) 限制因子家族成员（最著名的 A3G）的相互作用， 据报道，它们可以抑制 RT 的聚合酶活性，也将在项目 3 中进行研究。 除了慢病毒HIV-1的RTIC外，我们还将研究spumavirus原型泡沫病毒的RTIC （PFV）。我们正在采取多方面的方法来了解这些重要的病毒-宿主复合体。 相互作用，使用冷冻电镜、X 射线晶体学、HDX、SAXS 和预稳态动力学分析。 HDX 可解决的主要问题是： 与 DNA/DNA 相比，vRNA/tRNA 如何与 RT 相互作用 和 DNA/RNA 底物（冷冻电镜、X 射线、HDX、动力学） RNA 结构在该复合物中的作用是什么？ 它如何与 RT（冷冻电镜、X 射线、HDX）相互作用？引发的动态特性是什么？ HIV-1 和 PFV RTIC 中的相互作用有何不同？ HIV-1 RT 与 A3 蛋白相互作用以促进 HIV-1 限制？ HDX 对研究有帮助吗？ 基于喹啉的变构整合酶 (IN) 抑制剂 (ALLINI) 诱导全长野生型的异常多聚化 类型（WT）IN及其应用现在将扩展到检查最近发现的（核心4）噻吩- 基于抑制剂改变 IN 结构（项目 1）也将用于探测蛋白质间相互作用。 HIV-1 Gag 和 Gag-Pol 蛋白（项目 2）中，抗性突变或进化枝间差异的影响 HIV-1 蛋白中的蛋白间相互作用（项目 5），以及 HIV-1 蛋白与 SuFEx 或之间的相互作用 其他小分子探针（项目 6）将用于支持项目 6 和核心 4。