Reversible tuning of membrane sterol levels by cyclodextrin in a dialysis setting.
Biophysical journal – March 25, 2025
Source: PubMed
Summary
Researchers have developed a method to adjust sterol levels in liposomes, enhancing the study of membrane proteins. By using methyl-β-cyclodextrin in a controlled environment, they effectively manage cholesterol transfer. This reversible tuning improves membrane properties, paving the way for better insights into protein behavior and function.
Abstract
Large unilamellar vesicles are popular membrane models for studying the impact of lipids and bilayer properties on the structure and function of transmembrane proteins. However, the functional reconstitution of transmembrane proteins in liposomes can be challenging, especially, if the hydrophobic thickness of the protein does not match the thickness of the lipid bilayer. Such hydrophobic mismatch causes protein aggregation and low yields during the reconstitution procedure, which are exacerbated in sterol-rich membranes featuring low membrane compressibility. Here, we explore new approaches to reversibly tune the sterol content of (proteo)liposomes with methyl-β-cyclodextrin (mβCD) in a dialysis setting. Maintaining (proteo)liposomes in a confined compartment minimizes loss-of-material during cholesterol transfer and facilitates efficient removal of mβCD. We monitor the sterol concentration in the membrane with help of the solvatochromic probe C-Laurdan, which reports on lipid packing. Using Förster-resonance energy transfer, we show that cholesterol delivery to proteoliposomes induces the oligomerization of a membrane property sensor, while a subsequent removal of cholesterol demonstrates full reversibility. We propose that tuning membrane compressibility by mβCD-meditated cholesterol delivery and removal in a dialysis setup provides a new handle to study the impact of sterols and membrane compressibility on membrane protein structure, function, and dynamics.