Reversible tuning of membrane sterol levels by cyclodextrin in a dialysis setting

Abstract

Large unilamellar vesicles (LUVs) are popular membrane models for studying the impact of lipids and bilayer properties on the structure and function of individual membrane proteins. The functional reconstitution of transmembrane in liposomes can be challenging, especially, if the hydrophobic thickness of the protein does not match the thickness of the surrounding lipid bilayer. During the reconstitution procedure Such hydrophobic mismatch causes low yields and protein aggregation, which are exacerbated in sterol-rich membranes featuring low membrane compressibility. Here, we explore new approaches to reversibly tune membrane sterol contents proteoliposomes after their formation. Both cholesterol delivery and extraction are mediated by methyl-{beta}-cyclodextrin in a dialysis setting, which maintains (proteo)liposomes in a confined compartment. This makes it possible to reversibly tune the cholesterol level without losing membrane material simply by placing the dialysis cassette in a new bath containing either empty or cholesterol-loaded methyl-{beta}-cyclodextrin. Cholesterol delivery and removal is monitored with the solvatochromic probe C-Laurdan, which reports on lipid packing. Using Forster-resonance energy transfer, we show that cholesterol delivery to proteoliposomes induces the oligomerization of a membrane property sensor, while the subsequent removal of cholesterol demonstrates the full reversibility. We propose that tuning membrane compressibility by methyl-{beta}-cyclodextrin-meditated cholesterol delivery and removal in a dialysis setup provides a new handle to study its impact on membrane protein structure, function, and dynamics. Statement of significanceGenerating complex, sterol-rich, biomimetic membranes for studying the structure and function of reconstituted membrane proteins is challenging. As an important step towards asymmetric, sterol-rich, complex model membrane systems, we have established a procedure to control the membrane sterol level of liposomes and proteoliposomes using methyl-{beta}-cyclodextrin in a dialysis setup. We demonstrate the feasibility of this approach by C-Laurdan and dehydroergosterol fluorescence spectroscopy and gain control over the membrane sterol content. We explore several parameters that affect the rate of cholesterol delivery and show that the oligomerization of a membrane property sensor, which is on the unfolded protein response sensor protein Ire1, is controlled by the sterol content of the surrounding lipid bilayer.