title: Development and characterisation of novel therapeutic and biotechnological molecules
Creators: name: Allen, Nyasha
Publisher: name: University of Kent
Year: 2021

description: The cell membrane, made up of a complex arrangement of proteins and lipids, is an integral component of cells and functions as a protective barrier around cells. Interactions with cell membranes can impact the membrane dynamics, integrity and morphology and therefore have biological consequences. The study of these interactions allows a deeper understanding of fundamental biological processes, particularly the vast number of membrane related proteins whose physiological function is still currently unknown. This information may also allow us to determine the pathogenesis of diseases associated with these membrane related proteins. It can be utilised in the development of effective therapeutic agents against these diseases, such as bacterial infections, due to the significance of membranes in cell survival. Thus, this project is focused on understanding the interactions between biotechnological and therapeutic molecules with cell membranes through the use of synthetic liposomes, and a range of membrane binding assays. In the first part of this project, the antimicrobial activity of a series of membrane binding supramolecular self-associating amphiphiles (SSAs) was determined. Stepwise modifications were made to these molecules to ascertain their structure-activity relationships which were then utilised to produce second generation antimicrobials with improved E. coli activity. The anionic geometry of SSAs heavily influenced the acidity of the molecules and subsequent intermolecular interactions, which in turn impacted the antimicrobial activity observed. Lipid binding of SSAs was confirmed through the use of a competitive binding microscopy assay and fluorescence anisotropy experiments. The second-generation antimicrobials were found to gelate in salt solutions; thus, fluorescence microscopy was employed to characterise the gelation properties of these SSAs. In the second part of this study, the lipid binding properties of the membrane related protein alpha synuclein (α-Syn), which has been implicated in Parkinson's Disease (PD) was investigated. In addition, the impact of the post-translational modifications acetylation and phosphorylation was also explored. Unacetylated and Nt-acetylated α-Syn exhibited high affinities to CL and PE lipids in the thermal shift assays conducted. Further, fluorescence anisotropy binding experiments revealed binding of acetylated and unacetylated α-Syn to POPA, a key signalling lipid. Additionally, unacetylated α-Syn exhibited increased lipid binding activities in comparison to acetylated α-Syn, suggesting negative regulation of α-Syn by Nt-acetylation. The final part of this project was concerned with the lipid binding interactions of the C-elegans myosin 1 homolog, Hum-1, specifically its TH1 domain. Hum-1 TH1 was found to predominantly interact with anionic lipids, and phosphorylation of Hum-1 TH1 increased binding to PA lipids. This project demonstrated the value of investigating membrane lipid interactions in order to further understand the native roles and regulation of membrane proteins and improve the activity of membrane binding therapeutic molecules.