A diverse range of membrane peptides and proteins utilize membrane-curvature generation to carry out their biological function. Examples include cationic membrane peptides that disrupt microbial membranes or cross the membrane into cells by forming permanent or transient pores, and hydrophobic domains of viral fusion proteins that merge the virus envelope and the target cell membrane to cause viral entry. Elucidating how protein structures underlie membrane curvature generation has broad biomedical significance in enabling the design of more potent and resistance-free antibiotics and the development of new vaccines and antiviral drugs. The long-term objective of this project is to understand and quantify lipid-specific interactions of curvature-inducing membrane peptides. We will use solid-state NMR as our principal tool, because it is uniquely capable of simultaneously yielding high-resolution structures of membrane proteins and revealing the physical properties of the lipid membrane - curvature, dynamics, domain heterogeneity, and hydration - in which these peptides are embedded. We propose four specific aims. 1) We will investigate cationic-peptide- induced lipid clustering in bacteria-mimetic membranes. Potential segregation of anionic lipids from the main zwitterionic lipid of bacterial membranes, phosphatidylethanolamine, may be a key factor in promoting membrane curvature. Isotope-edited NMR experiments that probe lipid dynamics and peptide-lipid interactions will be conducted. Representative antimicrobial and cell-penetrating peptides such as PG-1 and HIV TAT will be examined. 2) We will develop 31P exchange NMR techniques to measure the curvature of mixed lipid membranes and the localization of cationic peptides in curvature-distinct domains. 3) We will determine the membrane-bound conformation, dynamics, depth of insertion, and oligomeric structure of the fusion peptide and the transmembrane domain of the paramyxovirus, PIV5. Structure information on PIV5 fusion protein will provide new insights into the mechanism of action of the important class I viral fusion proteins. 4) We will investigate the lipid interactions of the PIV5 fusion peptide using a variety of 2H, 31P, and 1H NMR experiments. Membrane curvature, fusion peptide localization, and membrane hydration will be characterized using both oriented and unoriented membranes, with the goal of understanding how the fusion peptide modifies the membrane structure to cause fusion.
Elucidating the structures and membrane interactions of antimicrobial peptides, cell-penetrating peptides, and viral fusion peptides may help the development of new resistance-free antibiotics, better drug-delivery agents, and new vaccines and antiviral drugs.
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