Solid State NMR Studies of the HIV-1 Fusion Peptide

HIV-1 融合肽的固态核磁共振研究

• 批准号: 6497375
• 负责人: David P Weliky
• 金额: $ 26.04万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6497375
• 关键词: HIV envelope protein gp41; cell fusion; circular dichroism; conformation; fluidity; host organism interaction; intermolecular interaction; membrane activity; membrane fusion; membrane model; molecular dynamics; molecular polarity; nuclear magnetic resonance spectroscopy; peptide analog; peptide chemical synthesis; peptide structure; protein protein interaction; protein structure function; radiotracer; structural biology; synthetic peptide; virus infection mechanism; virus receptors

The long-term goal of this research is to understand the atomic-level structural and dynamical basis for HIV-1 fusion peptide-induced membrane fusion. The peptide is derived from the N-terminal sequence of the HIV-1 gp41 envelope protein and is a critical domain for viral/host cell fusion. Numerous biochemical and biophysical studies have already shown that the free peptide is a biologically relevant model system for significant aspects of viral/host cell fusion including fusion peptide insertion and disruption of membranes. Hence, information provided by these new fusion peptide structural studies should be applicable to understanding the mechanism by which HIV-1 virions fuse with their target host cells. In addition, these studies should provide useful information for the general field of virus/host cell membrane fusion, which itself serves as a model for more physiologically beneficial cellular and vesicular fusion. During viral fusion, HIV-1 gp41 is believed to be trimerized such that the fusion peptide domains are in close proximity at their C-termini. In addition to structural studies on the membrane-bound 'monomeric' fusion peptide, intensive efforts will also be made to structurally characterize fusion peptides which have been synthetically trimerized. The main analytical tool in these studies is solid state NMR spectroscopy, a set of techniques which are well-suited to atomic-level structural studies in non-crystalline membrane systems. In conjunction with specific isotopic labeling, solid state NMR methods will be used to obtain the following types of residue-specific structural information about the membrane-bound fusion peptide: (1) conformation (secondary structure); (2) orientation relative to the membrane bilayer normal; and (3) oligomeric structures. The data from these three types of measurements will be combined to obtain a detailed picture of the membrane-bound fusion peptide structure. The experiments will employ a variety of solid state NMR methods including 2D MAS exchange and homo- and heteronuclear dipolar recoupling. An additional corollary aspect of the research is development of methods for preparation of peptide/membrane samples which provide biological relevance and are also suitable for solid state NMR spectroscopy. Fusion peptides will also be characterized in solution (prior to membrane insertion) using circular dichroism, gel filtration, light scattering, solution NMR spectroscopy, and analytical ultracentrifugation. Finally, 2H NMR will be used to probe local as well as global changes in lipid motional dynamics upon interaction with the fusion peptide. These structural and dynamical measurements should provide insight into the membrane-destabilizing effects of the fusion peptide which lead ultimately to membrane fusion.

本研究的长期目标是了解HIV-1融合肽诱导膜融合的原子水平结构和动力学基础。该肽衍生自HIV-1 gp 41包膜蛋白的N-末端序列，是病毒/宿主细胞融合的关键结构域。许多生物化学和生物物理学研究已经表明，游离肽是病毒/宿主细胞融合的重要方面的生物学相关模型系统，包括融合肽插入和膜破坏。因此，这些新的融合肽结构的研究提供的信息应适用于了解HIV-1病毒粒子与其靶宿主细胞融合的机制。此外，这些研究应该提供有用的信息，病毒/宿主细胞膜融合，这本身作为一个模型，更生理上有益的细胞和囊泡融合的一般领域。在病毒融合过程中，HIV-1 gp 41被认为是三聚化的，使得融合肽结构域在其C-末端非常接近。除了对膜结合的“单体”融合肽的结构研究之外，还将进行密集的努力来表征已经合成三聚化的融合肽的结构。这些研究中的主要分析工具是固态NMR光谱，这是一套非常适合于非结晶膜系统中原子级结构研究的技术。结合特异性同位素标记，固态NMR方法将用于获得关于膜结合融合肽的以下类型的残基特异性结构信息：（1）构象（二级结构）;（2）相对于膜双层法线的取向;和（3）寡聚结构。将这三种类型测量的数据合并，以获得膜结合融合肽结构的详细图像。实验将采用各种固态NMR方法，包括2D MAS交换和同质和异质偶极再耦合。该研究的另一个必然结果是开发用于制备肽/膜样品的方法，该方法提供生物相关性并且也适用于固态NMR光谱。融合肽还将在溶液中（在膜插入之前）使用圆二色性、凝胶过滤、光散射、溶液NMR光谱和分析超离心来表征。最后，2 H NMR将用于探测与融合肽相互作用后脂质运动动力学的局部和全局变化。这些结构和动力学的测量应该提供深入了解膜的融合肽，最终导致膜融合的不稳定的影响。