Biophysical and Structural Studies of Escherichia coli Mechanosensitive Channel of Large Conductance in Lipid Bilayers

Abstract

A major challenge of drug development is maximising specificity and efficacy at the lowest possible dose to minimise toxic side effects (Basile et al., 2012). Not only are the pharmaceutical windows small for both drug candidates and approved drugs alike, but they can also vary between individuals, making a dose level safe for one individual potentially harmful for another. One strategy for overcoming this problem is to develop new drug delivery methods where the drug is preferentially concentrated at the site of disease (Basile et al., 2012). In this respect, liposomal drug delivery systems have been used with success in topical applications in skin cancer treatment, and there is significant research interest in engineering similar systems for intravenous administration (Al-Jamal & Kostarelos, 2011;Fan & Zhang, 2013). Incorporating nanovalves into liposomes with controllable gating properties would further enhance the usefulness of liposomal drug delivery systems, providing a method to further regulate the release of liposome-encapsulated drugs from liposomes at the target site (Martinac et al., 2014).The mechanosensitive channel of large conductance (MscL) has been identified as a major candidate for the development of a protein-based nanovalve (Martinac et al., 2014). More generally, MscL is a prototype for the class of ion channels primarily gated by membrane tension, which includes medically significant proteins such as Piezo channels and TRP-type sodium channels (Martinac, 2011). As a well-expressing protein from Escherichia coli, MscL has been extensively studied both structurally and biophysically (Martinac, 2011). However, the channel gating mechanism of MscL, and by extension of mechanosensitive channels in general, remains poorly understood at the molecular level.This thesis aims to investigate the relationship between E. coli MscL and phospholipids both in the context of its structure and its behaviour in a lipid bilayer environment. While much has been studied on the channel gating properties of MscL in various phospholipid environments (Anishkin et al., 2005;Iscla et al., 2004;Iscla et al., 2011;Powl et al., 2008b;Tsai et al., 2005;Yoshimura et al., 2004), there remains a limited understanding of the behaviour and distribution of MscL in the bilayer. These are not only important from academic perspective as well as in the context of nanovalve development, but also for example, to identify factors which may limit the functional studies on MscL. More specifically, this thesis has focused on the identification of phospholipids closely associating with MscL, the distribution of MscL in phospholipid bilayers, and the incorporation of MscL into liposomes with heterogeneous phospholipids.The first research chapter (Chapter 2) outlines the optimisation of MscL expression and purification system for biophysical and structural studies. The original MscL construct had problems of ii heterogeneous expression and being not well-suited to reliable quantification by routine methods.New constructs were suc...