Monitoring detergent effects in lipid bilayers

Description

Reconstituting membrane proteins into lipid vesicles typically involves the use of detergent molecules. However, completely removing these detergent molecules from the system and detecting any residual molecules in the membrane can be difficult. Additionally, the impact of residual detergent on experiments, such as water permeability measurements, is unclear. To address this issue, we investigated the effect of residual octyl glucoside (OG) using the stopped-flow methodology. Thereby, large unilamellar vesicles from E.coli polar lipid extract with various concentrations of OG were challenged with a hyperosmotic gradient to induce water efflux from the vesicles. Analysis of time-dependent scattering and fluorescence self-quenching signals revealed that OG causes a maximal relative increase in membrane water permeability of 60-70% at room temperature, depending on the osmolyte used. Interestingly, in contrast to self-quenching experiments, an OGdependent second kinetic appeared in scattering mode. Using hyperosmotic shrinkage simulations, we found that the dilution of OG in the measurement cuvette during the application of the hyperosmotic buffer led to a temperature-dependent extraction of OG out of the lipid bilayer into the buffer. The kinetic was only visible in the scattering data due to its sensitivity to the refractive index of the vesicle defined by the refractive indices and the respective volume fractions of the membrane and the vesicle interior. The application of a hyperosmotic gradient did not
influence fluorescence self-quenching experiments as the process of detergent extraction, in turn decreasing the vesicle membrane area, only changes the vesicle shape towards a sphere but doesn't affect the volume change, which is solely defined by the osmotic gradient. It is unclear whether the rapid withdrawal of OG from the outer vesicle leaflet creates a mass imbalance between both leaflets, resulting in a estabilized inner leaflet forming mixed micellar structures within the inner monolayer, or if a flip-flop of OG between both monolayers occurs on a similar timescale as the detergent partitioning into the aqueous phase. Thus, stopped-flow light scattering experiments are a reliable method for monitoring detergent removal from lipid vesicles and proving the presence of detergent in vesicular membrane systems.

Period	07 Apr 2024 → 10 Apr 2024
Event title	Physical Modes of Action of Membrane-Active Compounds: 807. WE-Heraeus-Seminar
Event type	Seminar
Location	Bad Honnef, GermanyShow on map
Degree of Recognition	International