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: 10219107
• 负责人: Tatyana Polenova
• 金额: $ 30.2万
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10219107
• 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.

2 - MAS NMR技术开发计划摘要 已经清楚的是，构成HIV-1组装体的蛋白质分子内的动力学在HIV-1感染中起着关键作用。 调节病毒感染性，包括脱壳和成熟。该领域目前的进展受到以下因素的阻碍： 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，特别是当大幅度构象重排发生时。魔角 自旋（MAS）NMR是唯一定位，以产生这样的信息，当与分子结合时， 动力学（MD）模拟提供了任何其他手段都无法获得的动态信息。虽然MAS NMR是一种 虽然这是一种非常强大的技术，但它目前存在三个主要缺点：i）固有的低灵敏度，导致 测量时间长; ii）由于大量重叠信号，大型组件的光谱拥堵程度高;以及 iii）对大型系统进行广泛而耗时的数据分析（共振分配和结构 计算），阻碍了该方法的广泛使用。 我们建议开发一个新的方法论框架，用于原子级的结构，动态和机械 通过MAS NMR表征HIV-1蛋白质组装体，这将克服主要的障碍， 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，我们将整合高 超快MAS频率的磁场（17.6-28.2 T），质子探测，流线型数据采集， 处理和分析。我们还将采用基于动态核极化（DNP）的实验进行分析 低浓度的物种，以及调查的构象空间访问的动态 无序状态我们将开发适合于研究HIV-1蛋白质大型组装体的新实验， 它们与宿主蛋白质和小分子相互作用物的复合物，在一小部分时间内， 这是常规实验所需的材料。 通过组合可预见的显著灵敏度和/或分辨率增强 提出的MAS NMR和DNP方法将大大扩展系统的范围， 特征化通过实验和计算相结合的流线型数据分析， 大大提高了MAS NMR的吞吐量，并使该技术可用于非常广泛的人群， 研究人员通过拟议研究开发的用于分析HIV-1组装体的技术将 广泛适用于其他生物组件。最后，我们设想，拟议的方法框架 将为病毒蛋白质的原子分辨率结构和动力学表征铺平道路 完整的病毒。