In-situ Measurement of Cholesterol Transport in Model-Membrane Systems Studied by Time Resolve Small Angle Neutron Scattering and Comparison with MD Simulation

Abstract

Cholesterol is essential for a myriad of biological functions, but its excess is toxic. Cholesterol levels are maintained by various cholesterol metabolic pathways which depend critically on its intracellular transport and homeostasis. Any disorder in intracellular cholesterol distribution will lead to diseases, from neurodegenerative, such as Niemann Pick TYPE-C and Alzheimer, to cholelithiasis and atherosclerosis. Unfortunately, the understanding of intracellular transport of cholesterol has lagged behind other aspect of cholesterol metabolism due to limitations in the techniques used to date. These limitations have resulted in reported cholesterol transport rates showing huge inconsistencies. This is due to the fact that these measurements are not done in-situ, but rather require biochemical isolation of cholesterol-donor and cholesterol-acceptor vesicles. Another important limitation has been the need for a biochemical tag on cholesterol which has a significant effect on the resulting rates of transfer. This work presents in-situ Time-Resolved Small Angle Neutron Scattering (in-situ TR-SANS)studies of the Intra- and Inter-membrane cholesterol exchange rates in POPC model vesicles. This technique does not require any biochemical tag and transport of cholesterol from donor to acceptor vesicles can be measured continuously by following the changes in the scattering intensity. Interestingly our approach finds that trans-membrane flipping rates of cholesterol are much slower without any foreign particles in contrast to high flipping rates reported in literature. Molecular dynamics simulations have also been performed to investigate the energetic and kinetic behavior of cholesterol. We found that simulation results are in agreement with our SANS results, providing a more detailed thermodynamic description at the molecular level. Such a synergistic approach combining TR-SANS and MD simulation will provide new insight into the ongoing efforts of understanding cholesterol traffic and related disorders.