Technology Development 2: MAS NMR and dynamic nuclear polarization for HIV-1 structural biology

技术开发2：HIV-1结构生物学的MAS NMR和动态核极化

• 批准号: 9977963
• 负责人: Tatyana Polenova
• 金额: $ 22.84万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-27 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977963
• 关键词: Algorithms; Biological; Biology; Capsid; Cells; Complex; Congestive; Consumption; Data; Data Analyses; Detection; Disease; Electrons; Equipment; Event; Frequencies; Funding; Goals; HIV; HIV-1; Integration Host Factors; Investigation; Magic; Mechanics; Methodology; Methods; Molecular Conformation; Motion; Nuclear; Play; Positioning Attribute; Program Development; Protein Region; Proteins; Protocols documentation; Protons; Regulation; Research Personnel; Resolution; Resources; Role; Sampling; Signal Transduction; Structure; System; Techniques; Technology; Time; Viral; Viral Proteins; Virus; Virus-like particle; Work; base; cohort; data acquisition; detector; experimental study; improved; innovation; insight; instrumentation; magnetic field; molecular dynamics; novel; quantum; reconstruction; small molecule; structural biology; technology development; temporal measurement

Abstract for Technology Development Program 2 - MAS NMR It has become clear that dynamics within protein molecules comprising HIV-1 assemblies play critical roles in regulating viral infectivity, including uncoating and maturation. Current progress in the field is hampered by the paucity of structural-biological methods that yield atomic-level information into structure and dynamics simultaneously, particularly when large-amplitude conformational rearrangements take place. Magic angle spinning (MAS) NMR is uniquely positioned to yield such information and, when integrated with molecular dynamics (MD) simulations, provides dynamic information inaccessible by any other means. While MAS NMR is a very powerful technique, it currently suffers from three major drawbacks: i) inherent low sensitivity, resulting in long measurement times; ii) high spectral congestion for large assemblies due to numerous overlapping signals; and iii) extensive and time-consuming data analysis for large systems (resonance assignments and structure calculation), hampering the widespread use of the method. We propose to develop a new methodological framework for atomic-level structural, dynamic, and mechanistic characterization of HIV-1 protein assemblies by MAS NMR, which will overcome the main roadblocks, limited sensitivity and resolution, as well as long data analysis time. To accomplish this goal, we will integrate high magnetic fields (17.6–28.2 T) with ultrafast MAS frequencies, proton detection, streamlined data acquisition, processing, and analysis. We will also employ dynamic nuclear polarization (DNP)-based experiments for analysis of low-concentration species as well as for investigations of the conformational space accessible to the dynamically disordered states. We will develop new experiments suitable for studies of large assemblies of HIV-1 proteins and their complexes with host proteins and small-molecule interactors, in a fraction of time and with a small fraction of material that is required for conventional experiments. The dramatic sensitivity and/or resolution enhancements foreseen to become attainable through the combination of proposed MAS NMR and DNP methods will greatly expand the range of systems amenable to in-depth characterization. The streamlined data analysis through the integration of experiment and computation will dramatically improve the throughput of MAS NMR and make the technique accessible to a very wide cohort of researchers. The technologies developed for the analysis of HIV-1 assemblies through the proposed studies will be broadly applicable to other biological assemblies. Finally, we envision that the proposed methodological framework will pave the way for the atomic-resolution structural and dynamics characterization of viral proteins in the context of intact viruses.

技术开发计划摘要2-MAS核磁共振 很明显，组成HIV-1组件的蛋白质分子内的动力学在 调节病毒的感染性，包括脱壳和成熟。目前在这一领域的进展受到 缺乏将原子级信息转化为结构和动力学的结构生物学方法 同时，尤其是当发生大规模构象重排时。魔角 旋转(MAS)核磁共振是产生此类信息的独特位置，当与分子相结合时 动态(MD)模拟，提供任何其他方式无法访问的动态信息。而MAS核磁共振是一种 技术非常强大，但目前有三个主要缺点：i)固有的低灵敏度，导致 测量时间长；ii)由于许多重叠信号而导致大型组件的高频谱拥塞；以及 三)对大型系统进行广泛和耗时的数据分析(共振分配和结构 计算)，阻碍了该方法的广泛使用。 我们建议开发一种新的原子级结构、动态和机械的方法论框架 用MAS核磁共振表征HIV-1蛋白组件，这将克服主要障碍，有限 灵敏度和分辨率以及较长的数据分析时间。为了实现这一目标，我们将高度整合 具有超快MAS频率的磁场(17.6-28.2 T)、质子探测、简化的数据采集、 处理和分析。我们还将使用基于动态核极化(DNP)的实验进行分析 对低浓度物种的研究以及对动力学可及构象空间的研究 无序状态。我们将开发适合研究HIV-1蛋白大集合的新实验，并 它们与宿主蛋白质和小分子相互作用分子的络合物，在很短的时间内与一小部分 常规实验所需的材料。 预计将通过组合实现显著的灵敏度和/或分辨率增强 提出的MAS、核磁共振和DNP方法将极大地扩展适用于深入研究的系统的范围 人物刻画。通过实验和计算相结合的方式简化了数据分析 极大地提高了MAS核磁共振的吞吐量，并使该技术可供非常广泛的 研究人员。通过拟议的研究为分析艾滋病毒-1组件而开发的技术将是 广泛适用于其他生物组装体。最后，我们设想拟议的方法论框架 将为病毒蛋白的原子分辨结构和动力学表征铺平道路 完整的病毒。