Fusion of lipid membranes is an important natural process for the intra- and intercellular exchange of molecules. However, little is known about the actual fusion mechanism at the molecular level. In this study we examine a system that models the key features of this process. For the molecular recognition between opposing membranes two membrane anchored heterodimer coiled-coil forming peptides called 'E' (EIAALEK)3 and 'K' (KIAALKE)3 were used. Lipid monolayers and IR reflection absorption spectroscopy (IRRAS) revealed the interactions of the peptides 'E', 'K', and their parallel coiled-coil complex 'E/K' with the phospholipid membranes and thereby mimicked the pre- and postfusion states, respectively. The peptides adopted α-helical structures and were incorporated into the monolayers with parallel orientation. The strength of binding to the monolayer differed for the peptides and tethering them to the membrane increased the interactions even further. Remarkably, these interactions played a role even in the postfusion state. These findings shed light on important mechanistic details of the membrane fusion process in this model system. Furthermore, their implications will help to improve the rational design of new artificial membrane fusion systems, which have a wide range of potential applications in supramolecular chemistry and biomedicine.

A system based on two designed peptides, namely the cationic peptide K, (KIAALKE) 3 , and its complementary anionic counterpart called peptide E, (EIAALEK) 3 , has been used as a minimal model for membrane fusion, inspired by SNARE proteins. Although the fact that docking of separate vesicle populations via the formation of a dimeric E/K coiled-coil complex can be rationalized, the reasons for the peptides promoting fusion of vesicles cannot be fully explained. Therefore it is of significant interest to determine how the peptides aid in overcoming energetic barriers during lipid rearrangements leading to fusion. In this study, investigations of the peptides' interactions with neutral PC/PE/cholesterol membranes by fluorescence spectroscopy show that tryptophan-labeled K* binds to the membrane (K K*~6 .2 10 3 M À1 ), whereas E* remains fully water-solvated. 15 N-NMR spectroscopy, depth-dependent fluorescence quenching, CD-spectroscopy experiments, and MD simulations indicate a helix orientation of K* parallel to the membrane surface. Solid-state 31 P-NMR of oriented lipid membranes was used to study the impact of peptide incorporation on lipid headgroup alignment. The membrane-immersed K* is found to locally alter the bilayer curvature, accompanied by a change of headgroup orientation relative to the membrane normal and of the lipid composition in the vicinity of the bound peptide. The NMR results were supported by molecular dynamics simulations, which showed that K reorganizes the membrane composition in its vicinity, induces positive membrane curvature, and enhances the lipid tail protrusion probability. These effects are known to be fusion relevant. The combined results support the hypothesis for a twofold role of K in the mechanism of membrane fusion: 1) to bring opposing membranes into close proximity via coiled-coil formation and 2) to destabilize both membranes thereby promoting fusion.

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The designed coiled-coil-forming peptides E [(EIAALEK)3] and K [(KIAALKE)3] are known to trigger efficient membrane fusion when they are tethered to lipid vesicles in the form of lipopeptides. Knowledge of their secondary structure is a key element in understanding their role in membrane fusion. Special conditions can be found at the interface of the membrane, where the peptides are confined in close proximity to other peptide molecules as well as to the lipid interface. Consequently, different structural states were proposed for the peptides when tethered to this interface. Due to the multitude of possible states, determining the structure solely on the basis of circular dichroism (CD) spectra at a single temperature can be misleading. In addition, it has not yet been possible to unambiguously distinguish between the membrane-bound and the coiled-coil states of these peptides by means of infrared (IR) spectroscopy due to their very similar amide I' bands. Here, the molecular basis of this similarity is investigated by means of site-specific (13)C-labeled FTIR spectroscopy. Structural similarities between the membrane-interacting helix of K and the homo-coiled-coil-forming helix of E are shown to cause the similar spectroscopic properties. Furthermore, the peptide structure is investigated using temperature-dependent CD and IR spectroscopy, and it is shown that the different states can be distinguished on the basis of their thermal behavior. It is shown that the two peptides behave fundamentaly differently when tethered to the lipid membrane, which implies that their role during membrane fusion is different and the mechanism of this process is asymmetric.

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