SNARE derived peptide mimic inducing membrane fusionw

Membrane fusion is a central cellular process in eukaryotic cells. In the secretory pathway connecting organelles between the endoplasmic reticulum and the plasma membrane, fusion is mediated by sets of SNARE proteins ( soluble N-ethylmaleimide-sensitive factor attachment r eceptor). 1 Complementary sets of SNARE proteins are associated with the respective membranes. Upon contact, the SNAREs form a thermodynamically stable four helix bundle connecting the membranes (trans-complex) probably being generated in a zipper-like recognition process followed by helicalization. Thereby, the membranes are brought into proximity overcoming the energy barrier for membrane fusion. Neuronal exocytosis is catalyzed by the SNARE proteins SNAP-25 (two a-helices), VAMP2 (synaptobrevin), and syntaxin-1A (each one a-helix), with the two latter each possessing a single transmembrane domain (TMD) at the C-terminus (Fig. 1a). In vitro reconstitution of these SNAREs in liposomes ranging between 30–100 nm diameter results in SNARE-mediated membrane fusion. However, both assembly and fusion kinetics in these systems are highly complex; and it is difficult to differentiate between parameters affecting nucleation and zippering of the helix bundle and the downstream events of membrane contact, hemifusion, and fusion. In order to derive mechanistic insight into the fusion event, simpler model systems are needed. In particular, substitution of the SNARE complex by SNARE-mimicking peptides may shed light on the influence of the TMDs and the linker regions between the SNARE complex forming helices and the TMDs. In addition, investigation of various artificial recognition motifs opens the chance to investigate the molecular requirements fulfilled by SNARE complex formation. Several SNARE-mimicking model systems have been reported with the aim of reducing complexity in membrane fusion. Complementary artificial recognition motifs were linked to cholesterol, phospholipids, or membrane penetrating peptides and were subsequently embedded into the membrane. A variety of chemically diverse recognition motifs for linking the membranes has been reported such as the reversible reaction between boronic acid and diols, DNA double strands, and a vancomycin glycopeptide combined with a D-Ala-D-Ala moiety. Another reduced SNAREmodel has been introduced that is based on a coiled-coil forming a-peptide. Although in all of these cases specific membrane fusion was observed, they are structurally different from the SNAREs, and thus, do not allow for direct mechanistic extrapolation to SNARE-mediated fusion events. To overcome this limitation, we have recently introduced a new SNARE-mimicking model using the native TMDs linked to peptide nucleic acid (PNA) recognition motifs and obtained efficient fusion that proceeds via hemifusion as intermediate. We have now extended this approach by employing coiled-coil forming a-peptides. To this end, we have replaced the four SNARE helices (Fig. 1a) by smaller coiled-coil forming a-peptides (Fig. 1b and c). The hybrid between an a-peptide recognition motif and the SNARE derived TMDs is advantageous since it allows for studying the role of the native linkers and TMDs of synaptobrevin and syntaxin in the fusion reaction. Here, we report about the synthesis of these molecules and