Abstract
The most important aspect of synthetic lipid membrane architectures is their ability to study functional membrane-active peptides and membrane proteins in an environment close to nature. Here, we report on the generation and performance of a biomimetic platform, the S-layer supported lipid membrane (SsLM), to investigate the structural and electrical characteristics of the membrane-active peptide gramicidin and the transmembrane protein α-hemolysin in real-time using a quartz crystal microbalance with dissipation monitoring in combination with electrochemical impedance spectroscopy. A shift in membrane resistance is caused by the interaction of α-hemolysin and gramicidin with SsLMs, even if only an attachment onto, or functional channels through the lipid membrane, respectively, are formed. Moreover, the obtained results did not indicate the formation of functional α-hemolysin pores, but evidence for functional incorporation of gramicidin into this biomimetic architecture is provided.
Highlights
Biological protein pores and channel-forming peptides provide nanoscopic pathways for the flux of ions and other charged molecules across the hydrophobic portion of membranes in a variety of cells.These functions are essential for life and serve as key components in intercellular and intracellular communications [1,2] and represent very attractive drug targets
A solution of small unilamellar vesicles (SUVs) comprising of egg yolk phosphatidyl choline (Egg PC), 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine (DMPE; in both monolayers of the SUVs or only in the outer one), cholesterol and β-diketone ligand was passed over the SbpA lattice (Figure 1, step 3). This lipid was chosen for several reasons: the Egg PC is the bulk component forming the spherical vesicle; DMPE, as linker molecule is covalently bound with its terminal amino group to the activated SbpA; cholesterol has put in because it is the primary target for α-hemolysin [41] while, it attenuates modestly the action of gramicidin on phospholipid bilayers [42]
As no aperture is necessary to generate the S-layer supported lipid membrane (SsLM) in the Quartz crystal microbalance with dissipation monitoring (QCM-D) cell, combined
Summary
Biological protein pores and channel-forming peptides provide nanoscopic pathways for the flux of ions and other charged molecules across the hydrophobic portion of membranes in a variety of cells. In the present study, combined QCM-D and EIS measurements on one and the same preparation were used for the first time, for monitoring lipid membrane formation on the SbpA lattice and subsequent interaction with the channel-forming peptide gramicidin and the pore-forming protein α-hemolysin, respectively (Figure 1). Combining QCM-D and EIS to monitor structural changes and the formation of functional pores in the lipid membrane has recognized to constitute a straightforward approach to elucidate the mode of action and function of membrane-active peptides and membrane proteins [37] It became possible for the first time to generate SsLMs by a sophisticated vesicle fusion technique without the need of an aperture [28], which is a mandatory requirement for QCM-D measurements. This study constitutes the first attempt to study α-hemolysin and gramicidin incorporation in SsLMs directly generated within a QCM-D cell without the need of an aperture combined with EIS measurements on one and the same functionalized SsLM preparation
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