The influenza fusion peptide domain (HAfp23) is comprised of the N-terminal 23 residues of the hemagglutinin glycoprotein which are highly conserved and have been shown to be essential for membrane fusion and the infection process of the Influenza virus. HAfp23 has a helical hairpin structure consisting of two tightly packed amphiphilic helices that rest on the membrane surface. We reveal that HAfp23 is a new class of amphipathic helix that functions by leveraging the negative curvature induced by two tightly packed helices on membranes. The helical hairpin structure has an inverted wedge shape characteristic of negative curvature lipids, with a bulky hydrophobic region and a relatively small hydrophilic head region. The F3G mutation reduces this inverted wedge shape by reducing the volume of its hydrophobic base. We show that despite maintaining identical backbone structures and dynamics as the wild type HAfp23, the F3G mutant has an attenuated fusion activity that is correlated to its reduced ability to induce negative membrane curvature. The inverted wedge shape of HAfp23 is likely to play a crucial role in the initial stages of membrane fusion by stabilizing negative curvature in the fusion stalk.<br/><br/>Our investigation of the HAfp23 system led to our development of novel NMR techniques including Bicelle Induced Curvature and Sorting (BICS) and application of a new positively charged alignment media (Pinacyanol Acetate) for structure refinement. These methods were applied to the study of HAfp23 in combination with well-established NMR techniques, differential scanning calorimetry (DSC) and fluorescent lipid mixing assays. Furthermore, we have developed an NMR based approach to study HAfp23 in a more native lipid environment which we anticipate to be highly useful in the study of membrane proteins. The elucidations made on HAfp23 as well as the application of novel techniques to study membrane proteins are of high interest to the biophysical communities.

Influenza hemagglutinin fusion domain by advanced NMR using novel orthagonal refinement and BICS curvature measurements

253rd National Meeting (2017)

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