Tethered Bilayer Membranes as a Complementary Tool for Functional and Structural Studies: the Pyolysin Case
Giulio Preta, Marija Jankunec, Frank Heinrich, Sholeem Griffin, Iain Martin Sheldon, Gintaras Valincius
We demonstrate the use of tethered bilayers (tBLMs) as an experimental platform for functional and structural studies of membrane associated proteins by the electrochemical techniques. The reconstitution of the cholesterol-dependent cytolysin (CDC) pyolysin (PLO) from Trueperella pyogenes into tBLMs was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to PLO solutions were consistent with the dielectric barrier damage occurring through the formation of water-filled pores in membranes. Parallel experiments involving a mutant version of PLO, which is able to bind to the membranes but does not form oligomer pores, strengthens the reliability of this methodology, since no change in the electrochemical impedance was observed. Complementary atomic force microscopy (AFM) and neutron reflectometry (NR) measurements revealed structural details of the membrane bound PLO, consistent with the structural transformations of the membrane bound toxins found for other cholesterol dependent cytolysisn. In this work, using the tBLM platform we also observed a protective effect of the dynamin inhibitor Dynasore against pyolysin as well as pneumolysin. An effect of Dynasore in tBLMs, which was earlier observed in experiments with live cells, confirms the biological relevance of the tBLM models, as well as demonstrates the potential of the electrochemical impedance spectroscopy to quantify membrane damage by the pore forming toxins. In conclusion, tBLMs are a reliable and complementary method to explore the activity of CDCs in eukaryotic cells and to develop strategies to limit the toxic effects of CDCs.
membrane protein, neutron reflectometry, atomic force microscopy, bacterial lysin
, Jankunec, M.
, Heinrich, F.
, Griffin, S.
, Sheldon, I.
and Valincius, G.
Tethered Bilayer Membranes as a Complementary Tool for Functional and Structural Studies: the Pyolysin Case, Biochimica Et Biophysica Acta-Biomembranes, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=920512
(Accessed February 21, 2024)