Browsing by Author "Kumar, Pawan"

Membrane fusion is an essential process in nature and is often accomplished by the specific interaction of SNARE proteins. SNARE model systems, in which SNARE domains are replaced by small artificial units, represent valuable tools to study membrane fusion in vitro. The synthesis and analysis is presented of SNARE model peptides that exhibit a recognition motif composed of two different types of peptide nucleic acid (PNA) sequences. This novel recognition unit is designed to mimic the SNARE zippering mechanism that initiates SNARE-mediated fusion. It contains N-(2-aminoethyl)glycine-PNA (aeg-PNA) and alanyl-PNA, which both recognize the respective complementary strand but differ in duplex topology and duplex formation kinetics. The duplex formation of PNA hybrid oligomers as well as the fusogenicity of the model peptides in lipid-mixing assays were characterized and the peptides were found to induce liposome fusion. As an unexpected discovery, peptides with a recognition unit containing only five aeg-PNA nucleo amino acids were sufficient and most efficient to induce liposome fusion.

Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor proteins also known as SNAREs. The backbone of this essential process is the assembly of SNAREs from opposite membranes into tight four helix bundles forcing membranes in close proximity. With model systems resembling SNAREs with reduced complexity we aim to understand how these proteins work at the molecular level. Here, peptide nucleic acids (PNAs) are used as excellent candidates for mimicking the SNARE recognition motif by forming well-characterized duplex structures. Hybridization between complementary PNA strands anchored in liposomes through native transmembrane domains (TMDs) induces the merger of the outer leaflets of the participating vesicles but not of the inner leaflets. A series of PNA/peptide hybrids differing in the length of TMDs and charges at the C-terminal end is presented. Interestingly, mixing of both outer and inner leaflets is seen for TMDs containing an amide in place of the natural carboxylic acid at the C-terminal end. Charged side chains at the C-terminal end of the TMDs are shown to have a negative impact on the mixing of liposomes. The length of the TMDs is vital for fusion as with the use of shortened TMDs, fusion was completely prevented.

Fusion of lipid membranes to form a single bilayer is an essential process for life and provides important biological functions including neurotransmitter release. Membrane fusion proteins facilitate approximation of interacting membranes to overcome the energy barrier. In case of synaptic transmission, proteins involved are known as soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins. The SNAREs from synaptic vesicles interact with the SNAREs from the target membrane to form a coiled-coil bundle of four helices, thus pulling the membranes tightly together and initiating fusion. However, it remains unclear how these proteins function at molecular level. Natural systems are often too complex to obtain unambiguous results. Simple model systems mimicking natural proteins in synthetic lipid bilayers are powerful tools for obtaining insights into this essential biological process. An important advantage of such systems is their well-defined composition, which can be systematically varied in order to fully understand events at molecular level. In this review, selected model systems are presented based upon specific interactions between recognition units embedded in separate lipid bilayers mimicking native SNARE protein-mediated membrane fusion. Copyright (c) 2015 European Peptide Society and John Wiley & Sons, Ltd.