Int J Mol Sci. 2015 Jan 27;16(2):2824-38.
Probing peptide and protein insertion in a biomimetic S-layer supported lipid membrane platform.
Samar Damiati 1,2, Angelika Schrems 1,†, Eva-Kathrin Sinner 1, Uwe B. Sleytr 3 and Bernhard Schuster 1,*
1 Institute for Synthetic Bioarchitectures, Department of NanoBiotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, Vienna 1190, Austria; E-Mails: firstname.lastname@example.org (S.D.); email@example.com (A.S.); firstname.lastname@example.org (E.-K.S.)
2 Department of Biochemistry, King Abdulaziz University, Jeddah 21465, Saudi Arabia
3 Institute for Biophysics, Department of NanoBiotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, Vienna
† Present address: Sysmex Austria GmbH, Odoakergasse 34-36, Vienna 1160, Austria.
* Author to whom correspondence should be addressed; E-Mail: email@example.com; Tel.: +43-1-47654-80436
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.
Learning how Nature is organizing biomolecules by self-assembling to supramolecular architectures with unique features led to amazing concepts for the generation of new bio-inspired materials and functional surfaces. One prominent example is the cell envelope structure of bacteria and in particular archaea, which have evolved by Nature over billions of years (Fig. 1, A). The latter dwell under very extreme conditions like high pressure, temperature (up to 120 °C), and salt concentration and at a pH value down to zero. Surprisingly, the cell envelope structure of archaea is often simply composed of a lipid mono- (Fig. 1, A) or bilayer (Fig. 1, B) and an outermost protein lattice, which is in most cases anchored by hydrophobic domains in the lipid membrane (Fig. 1, A). These two-dimensional arrays of proteinaceous subunits forming surface layers on prokaryotic cells are defined as “S-layers”. We have studied the building principle of bacterial and archaeal cell envelope structures thoroughly in order to learn how to arrange in-vitro biomolecules like lipids and proteins on technologically relevant surfaces and interfaces like glass, silicon and titanium oxide, metals (gold, platinum, stainless steel, etc.) and polymers (Fig. 1, B).
Figure 1: A): Sketch of a supramolecular structure of an archaeal cell envelope architecture comprising a cytoplasmic membrane (composed of tetraether lipids; grey), archaeal S-layer proteins (yellow), which are anchored in the lipidic matrix and integral membrane proteins (violet). B): Schematic drawing of a solid support (sensor or electrode; brown) where a closed bacterial S-layer lattice (yellow) has been assembled. On this proteinaceous structure, a phospholipid membrane (grey with violet head groups) was generated. Finally, this biomimetic membrane can be functionalized by the reconstitution of integral membrane proteins (green), e.g., a selective ion channel permitting the passage of specific ions (red). Not drawn to scale.
The challenge of integrating non-living systems with biological ones lead to problems associated with material interfacing and compatibility, as well as biological issues, such as viability and stability. Furthermore, water is necessary to maintain biological units, but has adverse effects on engineering components. Bottom-up and self-assembly are the most common strategies in nanobiotechnology used for the organization of biological units. The uprating of this strategy to the creation of hybrid devices is reckoned with distinguished prospects. Our challenge was to form smart, stable and viable biomimetic membranes, which also permit the proper incorporation of biological elements based on concepts, ideas and inspiration from biological systems. Finally, we were successful in assembling these artificial membrane platforms on sensor surfaces and electrodes. The innovative novelty is the S-layer lattice in-between the inorganic support and the lipid membrane. This protein layer acts as a stabilizing and anchoring scaffold, provides a biological cushion covering the inorganic support and constitutes an ionic reservoir and antifouling surface. Moreover, it facilitates the lateral diffusion of lipids within the membrane compared with other cushion materials and the incorporation of integral membrane proteins having bulky domains protruding from the attached lipid membrane. This striking achievement allows the detailed investigation of the structure and function of the membrane itself but also of reconstituted membrane-active peptides and trans-membrane proteins by means of advanced microscopical and surface-sensitive techniques.
Membrane proteins as amphiphilic molecules possess complex refolding processes that allow them to attain the three-dimensional configuration and thus, restoration of functionality only in lipid membranes. Moreover, these proteins are of major interest in medicine, diagnostics and the pharmaceutical industry. Approximately 60% of all presently known over 430 drug targets are membrane proteins, which are involved with cell sensing, signal transduction, immune recognition, transport of ions and nutrients, and a host of other vital process and thus, in health and disease. Among this 60%, G-protein coupled receptors are most prevalent (19%), followed by ion channels (17%), receptors (13%) and membrane-associated enzymes (6%). Moreover, the results from the human proteome project suggest that more than 30% of proteins are membrane or membrane-associated proteins like pores, ion channels, membrane-anchored enzymes, and most important (G-protein coupled) receptors. However, investigations on these proteins pose significant technical challenges, primarily due to their physico-chemical structure and sensitivity outside their native environment. They may contain large hydrophobic moieties which anchor them in the membrane but as a consequence their solubility is very limited in most biological media. For this reason, biomimetic model lipid membranes have attracted lively interest because of their unique feature to provide an amphiphilic matrix for reconstitution of (trans-)membrane proteins. Hence, membrane proteins as preferred targets for pharmaceuticals reconstituted in versatile lipid membrane platforms received widespread recognition in drug discovery and protein-ligand screening, and are of high interest for the development of biosensors.
A great deal of literature can be found for the membrane-active peptides alamethicin, valinomycin and gramicidin. The latter was also used to determine the fluidity of the lipid membrane and as molecular force probe. In general, several naturally derived peptides have been figured out to constitute successful drugs for many years. However, in recent years antimicrobial peptides gained in interest as it turned out that global antibacterial resistance is becoming an increasing public health problem. One important task in this respect is to elucidate the mode of action of antimicrobial peptides in order to use them as therapeutics. Despite the potential obstacles that remain, therapeutic peptides are prominent candidates for playing a significant role in the treatment of diseases ranging from Alzheimer’s disease to cancer.
The lipid membrane platform developed in our group could so far be successfully utilized to elucidate insertion and the mode of action of several membrane-active and antimicrobial peptides. Moreover, we have already presented an elegant synthesis and reconstitution approach for functional studies on voltage responsive membrane proteins. In this feasibility study, we focused on the functional reconstitution of the voltage-dependent anion channel (VDAC) from human mitochondria into the planar architecture of an S-layer-supported lipid membrane (SsLM). The chosen cell-free protein synthesis (CFPS) strategy (Fig. 2), in which VDAC proteins are synthesized in bacterial cell lysate in the presence of a lipid membrane platform offers a great advantage over the conventional, cell-based synthesis. First, with the CFPS method the complex cell culture, expression and purification steps can be circumvented and second, the protein of interest has not to be solubilized in a detergent. Moreover, the challenges of investigating biological systems, such as membrane proteins, can be lightened by re-designing and mimicking existing, natural membrane architectures using synthetic alternatives. Thus, physiological investigations of subtle membrane proteins, such as the VDAC protein species, can be achieved by the combination of CFPS and SsLMs as recently reported by us.
Figure 2: Schematic flow chart showing the cell-free protein synthesis (CFPS) of the human voltage-dependent anion channel (VDAC) and subsequent insertion into the S-layer supported lipid membrane (SsLM). The nascent polypeptide chain can immediately insert into the present SsLM and hence, is able to adopt its functional structure by proper folding in the lipid matrix.
By using highly sensitive biophysical techniques such as quartz crystal microbalance with dissipation monitoring and electrochemical impedance spectroscopy, the VDAC protein could be studied in a robust environment, the SsLM. This illustrates the potential of our approach for developing a biosynthetic architecture with engineered properties that promotes stability and reproducibility, and at the same time permits access to the family of membrane proteins being notoriously difficult to handle in the complexity of a living cell. The capabilities of supported lipid membranes to be functionalized by membrane-active (antimicrobial) peptides or membrane proteins either detergent-solubilized or particularly by the CFPS have opened the door to biotechnology applications in medicine, diagnostics, pharmacy, biosensor systems, and environmental monitoring.
 Damiati, S., Schrems, A., Sinner, E.K., Sleytr, U.B., Schuster, B. 2015. Probing peptide and protein insertion in a biomimetic S-layer supported lipid membrane platform. Int. J. Mol. Sci. 16: 2824-2838.
 Damiati S., Zayni S., Schrems A., Kiene E., Sleytr U.B., Chopineau J., Schuster B., Sinner, E.K. 2015 Inspired and stabilized by nature: Ribosomal synthesis of the human voltage gated ion channel (VDAC) into 2D-protein-tethered lipid interfaces. Biomater. Sci. 3: 1406-1413.
 Schuster, B. & Sleytr, U.B. 2015. Relevance of glycosylation of S-layer proteins for cell surface properties. Acta Biomater. 19: 149-157.
 Schuster, B. & Sleytr, U.B. 2015. Biomimetic S-layer – lipid self-assemblies as platform for membrane-active peptides and proteins. Eur. Biophys. J. Biophys. 44 (1): S98.
 Schuster B. & Sleytr, U.B. 2014. Biomimetic interfaces comprised of S-layer proteins, lipid membranes and membrane proteins. J. R. Soc. Interface 11: 20140232.
 Schrems, A., Larisch, V.-D., Sleytr, U.B., Hohenegger, M., Lohner, K., Schuster, B. 2013. Insertion of an anionic analogue of the antimicrobial peptide PGLa in lipid architectures including S-layer supported lipid bilayers. Curr. Nanosci. 9: 262-270.
 Schrems, A., Larisch, V.-D., Stanetty, C., Dutter, K., Damiati, S., Sleytr, U.B., Schuster, B. 2011. Liposome fusion on proteinaceous S-layer lattices triggered via β-diketone ligand – europium(III) complex formation. Soft Matter 7: 5514-5518.
 Schrems, A., Kibrom, A., Küpcü, S., Kiene, E., Sleytr, U.B., Schuster, B. 2011. Bilayer lipid membrane formation on a chemically modified S-layer lattice. Langmuir 27:3731-3738.
Bernhard Schuster, PhD
Institute for Synthetic Bioarchitectures
Department of NanoBiotechnology
BOKU – University of Natural Resources and Life Sciences, Vienna
Muthgasse 11, 1190 Vienna, Austria.
Phone number: +43-1-47654-80436
Email address: firstname.lastname@example.org