Solid State NMR Studies of the HIV-1 Fusion Peptide

这项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。该肽源自 HIV-1 gp41 包膜蛋白的 N 端序列，是病毒/宿主细胞融合的关键结构域。大量的生化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研究提供的信息应该适用于理解 HIV-1 病毒体与其靶宿主细胞融合的机制。此外，这些研究应该为病毒/宿主细胞膜融合的一般领域提供有用的信息，病毒/宿主细胞膜融合本身可以作为生理上更有益的细胞和囊泡融合的模型。在病毒融合过程中，HIV-1 gp41被认为是三聚化的，使得融合肽结构域在它们的C末端非常接近。除了膜结合“单体”融合肽的结构研究外，还将大力研究合成三聚化的融合肽的结构特征。这些研究中的主要分析工具是固态核磁共振波谱，这是一套非常适合非晶膜系统原子级结构研究的技术。结合特定同位素标记，固态NMR方法将用于获得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方向； (3)寡聚结构。来自这三种类型的测量的数据将被组合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核磁共振方法，包括 2D MAS 交换以及同核和异核偶极重偶联。该研究的另一个必然方面是开发肽/膜样品的制备方法，该方法提供生物相关性并且也适用于固态核磁共振波谱。还将使用圆二色性、凝胶过滤、光散射、溶液核磁共振光谱和分析超速离心对溶液中的融合肽进行表征（在膜插入之前）。最后，2H NMR 将用于探测与融合肽相互作用时脂质运动动力学的局部和整体变化。这些结构和动力学测量应该可以深入了解融合肽的膜不稳定效应，最终导致膜融合。

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.