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.
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