Engineering artificial protein-protein interactions through membranes with controllable architectures
Cell membranes are a complex mixture of lipids and proteins, all of which vary in size, shape and composition. The ability for a cell to control its membrane, and regulate its contents, is in large part due to the regulation of local composition, curvature and tension. These changes to membrane properties can in turn alter the behaviour of membrane bound proteins. Understanding how these membranes, and their fundamental properties, can influence proteins in vitro is essential for building true
... ynthetic cells, and building artificial cellular networks. In this thesis, work is presented which investigates the effect of membrane composition on the activity of the mechanosensitive channel of large conductance (MscL) from E.Coli. Using different model membrane systems, and many activation mechanisms of the protein, a greater understanding of MscL function has been gained. The first reconstitution, and activity measurement, of MscL into droplet interface bilayers (DIBs) is presented. Being able to measure MscL activity in the DIB model membrane has then lead to whole new avenues of research. The DIB system allowed for electrophysiological recording of single MscL channels, exploring the ability to see in-depth changes to MscL activity as DIB composition changes. Then, by linking multiple DIBs together, a network of bilayers connected by MscL channels has been presented. The first time a synthetic bilayer network linked by a gated ion channel has been demonstrated. Finally, a new device has been built allowing the generation of a measurable amount of shear force inside a DIB, and MscL activity was measured. It is hoped that the results of this thesis, will lead to further studies of more complex biological systems, deeper mechanistic studies of membrane proteins, and further synthetic networks of membrane protein interactions.