Using vesicles to study the effect of sterols on the mechanical strength of lipid membranes and the protein-lipid membrane Interaction

In this thesis, studies of two biophysical topics will be discussed: the effect of sterols on the mechanical strength of lipid membranes, and the interaction between Cytidine 5'-triphosphate(CTP):phosphocholine cytidylyltransferase (CCT) and lipid membranes. The mechanical strength of lipid membranes was probed by measuring the lysis tension of vesicles, as determined from the minimum pressure required to extrude vesicles through small pores. The vesicles used in these experiments were made from mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and various sterols, including cholesterol, lanosterol and ergosterol. The effect of the sterol concentration on the lysis tension was determined. The results show that all sterols increase the lysis tension of POPC membranes, where cholesterol shows the largest effect followed by lanosterol and ergosterol. The increase in the lysis tension of POPC membranes by sterols is correlated to the increase in the chain order of the lipids by sterols. The increase in the strength of lipid membranes by sterols indicates their contribution to cell viability, which depends on maintaining an intact plasma membrane. The interaction between CCT and lipid membranes was studied by observing vesicle aggregation induced by CCT. This was conducted by measuring the size and polydispersity of vesicles before and after the introduction of CCT using dynamic light scattering. Vesicles for these studies were made both from lipids that activate CCT (activating lipids) and lipids that do not activate CCT (non-activating lipids). The activating lipids investigated include anionic lipids (class I lipids) and lipids that produce negative curvature in membrane (class II lipids). Aggregation occurs when CCT is introduced to samples of class I lipid vesicles. In contrast, there is no indication of aggregation when CCT is introduced to samples of vesicles made from both non-activating and class II lipids. The occurrence of aggregation depends on the binding strength of CCT to the membrane. The results suggest that CCT cross-bridges two vesicles, which is a new aspect of CCT interaction with lipid membranes.