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.

摘要 蛋白质是动态的分子，经历不断的弯曲和运动。蛋白质构象的变化 和动力学对分子识别很重要，这些变化发生在许多关键的生物学过程中 包括酶的激活和抑制、与受体的配基结合、翻译后 修饰，以及跨蛋白质-蛋白质界面的变构通讯。所有这些过程都是 通过功能复合体内蛋白质的结构和动态进行调节。因此，一些技术可以 研究蛋白质的结构和动力学对于理解生物过程和帮助 开发针对疾病治疗干预的蛋白质的策略。HDX-核磁共振核心将解决 蜂巢中心的中心目标包括表征艾滋病毒蛋白质之间的相互作用， 用宿主分子，或用小分子探针。HDX工作的一个关键重点将是结构和动态 HIV-1逆转录酶(RT)起始复合体，或RTIC，一个大型的蛋白质-核酸复合体 包括RT(p66/p51异源二聚体)、5‘未翻译病毒RNA(VRNA)的引物结合部位区域和 一个人的tRNA(项目3：Arnold，Musier-Forsyth，Griffin，Lyumkis和Sarafianos)。我们还将调查 HIV-1RT与APOBEC3(A3)限制因子家族成员(尤其是A3G)的相互作用， 它们已经被报道抑制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理解这些重要的病毒-宿主复合体 相互作用，使用冷冻-EM、X射线结晶学、HDX、SAXS和稳态前动力学分析。其中 HDX可以解决的主要问题是：与DNA/DNA相比，vRNA/tRNA如何与RT相互作用 和DNA/RNA底物(冷冻-EM、X射线、HDX、动力学)？RNA结构在这个复合体中的作用是什么？ 它是如何与RT(冷冻-EM、X射线、HDX)相互作用的？起爆的动力特性是什么？ 复杂(冷冻-EM、SAXS、HDX、动力学)？HIV-1和PFV RTIC之间的相互作用有何不同？何以 HIV-1逆转录酶与A3蛋白相互作用促进HIV-1限制？HDX一直是研究的工具 喹啉类别构整合酶(IN)抑制剂(ALLINI)诱导野生型全长野生型细胞异常多聚体 (WT)IN类型及其应用现在将扩展到检查最近发现的(核心4)噻吩烯- 碱性抑制剂改变IN的结构(项目1)。HDX-MS还将用于探测蛋白质间的相互作用 在HIV-1 Gag和Gag-Pol蛋白(项目2)中，耐药突变或分支间差异的影响 HIV-1蛋白之间的相互作用(项目5)，以及HIV-1蛋白与SuFEx或SuFEx之间的相互作用 其他小分子探针(项目6)。核磁共振将用于支持项目6和核心4。