Surface Plasmon Resonance Spectroscopic Study on Pore-Forming Behavior of Streptolysin O on Supported Phospholipid Bilay
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Surface Plasmon Resonance Spectroscopic Study on Pore-Forming Behavior of Streptolysin O on Supported Phospholipid Bilayers Thomas Wilkop, Danke Xu and Quan Cheng* Department of Chemistry University of California Riverside, California 92521 Corresponding author: [email protected] Tel: (909) 787-2702 Abstract A novel supported bilayer membrane was formed on top of a hydrophobic spacer layer of hexyl thioctate (HT) and its application in the detection of pore forming toxins was demonstrated. The formation kinetics, effect of different flow rates on the fusion process of vesicles and perforation by incubation with streptolysin O (SLO) and pristine membranes were investigated. Individual layers of the multilayer structure were successfully characterized with surface plasmon resonance (SPR), and the resulting spectra were analyzed in order to determine the layer thickness and other membrane properties. The interaction of SLO with the pristine biomimetic membrane resulted in a pronounced change in the reflectivity spectrum. Elements of these spectral changes were analyzed to explain the process of toxin binding and pore formation taking place on the lipid membrane. Introduction Pore-forming toxins (PFTs) exert their cytotoxicity by permeabilizing the plasma membrane of cells1, which in turn leads to a loss of intracellular enzymes and essential content. This destroys the osmotic cell balance culminating in cell death. Streptolysin O (SLO) is a 61 kDa PFT 2 produced by Streptococcus pyogenes A and C which attaches to cholesterol (CHO) in the target membranes. Several SLO molecules associate with each other to form arc-and ringshaped clusters that insert into the bilayer to produce oligomeric transmembrane pores of up to 35 nm in diameter3. Pore-formation by SLO can be observed in erythrocyte membranes and CHO-rich bilayer vesicles4. The latter opens up possibilities to design biomimetic membrane systems in which the membrane-mediated flux can be determined by the presence or absence of the SLO. Recently interest in the use of toxin biomimetic membrane interactions 5,6 has surged. In order to develop a successful sensor utilizing supported bilayers with a modulated permeability, various influences and steps in the perforation forming task have to be well understood. The process by which phospholipid molecules attach themselves to a hydrophobic monolayer can be described as follows. Lipid vesicles diffuse to the hydrophobic surface in a process that is rate limiting7 while the fusing and reorganization of the lipid is fast. In the investigated system two types of vesicles fuse onto the hydrophobic layer, a) intact vesicles and b) vesicles that have already undergone incubation with pore forming SLO, hence having a perforated shell.
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Surface plasmon resonance spect
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