Investigating the Role of Temperature and Surfactant Saturation in Triton X-100 Mediated Proteoliposome Production

Reconstituting membrane proteins into liposomes to form proteoliposomes is an essential tool for studying structural and functional properties of these biomolecules. Detergent-mediated reconsti-tution is a well-established method of inserting these membrane-anchored proteins into liposome membranes, but limited data has been published investigating the role of temperature in this pro-cess. In this study, the temperature at which multicomponent liposomes are saturated with Triton X-100, a commonly used nonionic detergent, was manipulated during the reconstitution protocol of a model protein, SARS-CoV-2 Spike glycoprotein. Several parameters were monitored across different saturation temperatures: the concentration of detergent required to saturate these lipo-somes, the amount of polystyrene adsorbent "BioBeads" required to remove the detergent from the liposomes, and the physical properties of resulting particles, including the average number of pro-teins inserted in each proteoliposome. The temperature-dependence of Triton X-100 saturation of the liposomes investigated here was found to be minimal beneath 60 ?C, and a saturation ratio range of 4.6-9.2 moles Triton X-100 per mole of lipid was required to saturate these multicompo-nent particles. Removal of Triton X-100 by BioBeads was similarly unaffected by the saturation temperature. Importantly, the cloud point behaviour of Triton X-100 was highly implicated in the aggregation of liposomes and proteoliposomes. Localization of proteins within particle-aggregate dispersions suggests that while saturation temperature does not seem to influence the density of proteins reconstituted in each proteoliposome, at elevated temperatures the aggregation effect re-duces the total yield of proteoliposomes. The approach presented here provides a framework for improving and assessing detergent-mediated protein reconstitution processes and suggests key factors to consider for experimental design of proteoliposome preparation.