Drug-Membrane and Protein Interactions in tethered Bilayer Lipid Membranes Studied by Surface Analytical Methods

Author: Julius Zieleniecki

Zieleniecki, Julius, 2017 Drug-Membrane and Protein Interactions in tethered Bilayer Lipid Membranes Studied by Surface Analytical Methods, Flinders University, School of Chemical and Physical Sciences

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Approximately 60–70% of all drugs released in 2006-2008 were designed specifically to target membrane bound proteins [1-3]. Although these drugs are targeted, they also interact non-specifically; which is suggested to be one cause of their side-effects. Even though lipid membranes are one of the major building blocks of a cell as well as the organelles within them, they are currently significantly understudied. To improve how drugs interact with the body, the properties of membranes as well as molecules interacting with them should be studied further. In this case, focusing on the simple anaesthetic ethanol, a literature search indicates that few studies have set out to understand how ethanol interacts with a simple, protein or peptide-tested, and realistic, model membrane both electrically as well as physically. Equally understudied as membranes and non-specific drug interactions, are cellularmembrane proteins. These proteins are difficult to crystallise and generally embedded deep within a cell, unreachable to study. Although computational methods and speeds are improving, being able to reconstitute these proteins without the need for bacteria, directly from RNA into an in vitro membrane which can be studied both electrically as well as physical, affords unprecedented improvements in supporting computational studies as well as learning about these proteins. This thesis is a summary of experiments undertaken regarding: 1) the interactions of ethanol with a tethered Bilayer Lipid Membrane (tBLM) and 2) the use of a tBLM as a matrix for embedding and testing the protein HvBot1 translated in vitro by cell-free synthesis. In the first series of experiments, tBLMs were tested using Electrochemical Impedance Spectroscopy (EIS), Femto-Second Vibrational Sum Frequency Generation (fs-vSFG), andNeutron Reflectometry (NR) to interpret: the electronic and physical effect of ethanol on a tBLM; the position of ethanol within a tBLM; and the indirect effect of ethanol on the peptide Valinomycin within the membrane at various ethanol concentrations. In the second series of experiments the same type of tBLM system was used, in conjunction with cell-free synthesis, to observe and test the expression and embedding of HvBot1 formed from RNA in vitro in the tBLM system. Overall this research develops the knowledge of membrane-drug interactions and extends the understanding of the viability of cell-free synthesis of large proteins such as HvBot1 with tBLMs.

Keywords: Flinders, Nanotechnology, Bio-nanotechnology, surface science, tethered bilayer lipid membranes, tethered membranes, tBLMs, drug interactions, in vitro, impedance spectroscopy, femto-second, sum frequency generation, Neutron Reflectometry, Valinomycin, DPhyTL, DPhyPC, Phoschocholine, cell-free synthesis, HvBot1, borate transporter
Subject: Chemistry thesis

Thesis type: Doctor of Philosophy
Completed: 2017
School: School of Chemical and Physical Sciences
Supervisor: Dr. Ingo Koeper