Influenza Fusion Mechanism Figure 10. Detailed view of the ‘super’ bundle (10 ms) formed by the leaky fusion mutant G13A (Top view, cross-section through the bilayer center). Notice that residue 13 (colored green) directly faces the hydrophobic lipid rim around the bundle. The solvent (colored blue) in the center of the bundle suggests the occurrence of leakage prior to lipid mixing [11]. doi:10.1371/journal.pone.0038302.g010 head-group plane five times more often per time interval. If these protrusions would enhance stalk formation directly via the formation of splayed lipid intermediates, they are expected to lower the apparent stalk barrier by, ln(5)kBT = 1.6 kBT [46]. Notably, a larger peptide:lipid ratio likely enhances such effect [67]. However, because of the logarithmic dependence, in order to lower the stalk barrier, by e.g. 15–40 kBT, one would require an 106{1017 fold increase in the total protrusion frequency. Thus, it seems rather unlikely that the wild-type peptide facilitates stalk formation directly via tail protrusions. In addition, it is hard to reconcile how protrusions, when only being involved in stalk formation, would explain the action of the terminal hemifusion mutants G1S/E11A and the leaky fusion mutant G13A. Tail protrusions, or equivalently membrane thinning, reflect the presence of stress. Apparently, the adsorption of the wild-type peptide induces a substantial stress in the membrane. For a surface adhered amphiphilic peptide, however, the ability to induce stress is limited because the membrane will alternatively respond by forming pores or trans-membrane arranged bundles (i.e., when the peptides are lineactants) [50,51]. In addition, such building up of surface stress causes competitive bending of the membrane [68]. Thus, the peptide-induced stress, i.e. the observed protrusions/ thinning, might also facilitate other processes such as bundle, pore and dimple formation [7,52]. With the dimple being hypothesized as a highly fusogenic and thus very transient intermediate in membrane fusion, one would not expect its direct observation. However, the observation of (intact) dimples by cryo-electron microscopy in wild-type influenza fusion [7] suggests that these dimples are in fact long-lived states with a slow escape rate – they are quite resistant to fusion. We PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e38302