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: 6332121
• 负责人: David P Weliky
• 金额: $ 14.19万
• 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/6332121
• 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.

这项研究的长期目标是了解HIV-1融合肽诱导的膜融合的原子水平结构和动力学基础。该肽源自HIV-1 GP41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。许多生化和生物物理研究已经表明，游离肽是一种与生物学相关的模型系统，用于病毒/宿主细胞融合的重要方面，包括融合肽插入和膜的破坏。因此，这些新的融合肽结构研究提供的信息应适用于了解HIV-1病毒体与目标宿主细胞融合的机制。此外，这些研究应为病毒/宿主细胞膜融合的一般领域提供有用的信息，该病毒/宿主细胞膜融合本身是一种模型，用于更有益的细胞和囊泡融合。在病毒融合过程中，HIV-1 GP41被认为是对融合肽结构域在其C末端处紧密近端的修剪度。除了对膜结合的“单体”融合肽进行结构研究外，还将在结构上进行大量努力，以在结构上表征已通过合成修剪的融合肽。这些研究中的主要分析工具是固态NMR光谱，这是一组非常适合非晶体膜系统原子级结构研究的技术。结合特定的同位素标记，将使用固态NMR方法来获得有关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）相对于膜双层正常的方向； （3）寡聚结构。这三种类型的测量结果将合并，以获得膜结合的融合肽结构的详细图片。该实验将采用多种固态NMR方法，包括2D MAS交换以及同型和异核偶极回合。该研究的另一个推论方面是开发用于制备肽/膜样品的方法，这些方法具有生物学相关性，也适用于固态NMR光谱。融合肽也将在溶液（膜插入之前）使用圆形二分性，凝胶过滤，光散射，溶液NMR光谱和分析性超速离心。最后，与融合肽相互作用后，2H NMR将用于探测脂质运动动力学的局部变化和全局变化。这些结构和动力学测量应为融合肽的膜 - 二动作用提供见解，这最终导致膜融合。