Alsayyah Cynthia, Rodrigues Emmanuel, Hach Julia, Renne Mike F, Ernst Robert
Medical Biochemistry and Molecular Biology, Medical Faculty, Saarland University, Homburg, Saar, Germany; Preclinical Center for Molecular Signaling (PZMS), Medical Faculty, Saarland University, Homburg, Saar, Germany; Center for Biophysics (ZBP), Saarland University, Saarland, Germany.
Medical Biochemistry and Molecular Biology, Medical Faculty, Saarland University, Homburg, Saar, Germany; Preclinical Center for Molecular Signaling (PZMS), Medical Faculty, Saarland University, Homburg, Saar, Germany; Center for Biophysics (ZBP), Saarland University, Saarland, Germany.
Biophys J. 2025 May 6;124(9):1433-1445. doi: 10.1016/j.bpj.2025.03.020. Epub 2025 Mar 25.
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, whereas 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.
大单层囊泡是用于研究脂质和双层特性对跨膜蛋白结构和功能影响的常用膜模型。然而，跨膜蛋白在脂质体中的功能重建可能具有挑战性，特别是当蛋白质的疏水厚度与脂质双层的厚度不匹配时。这种疏水不匹配会导致蛋白质聚集和重建过程中的低产率，在具有低膜压缩性的富含甾醇的膜中这种情况会更加严重。在这里，我们探索了在透析环境中用甲基-β-环糊精（mβCD）可逆调节（蛋白）脂质体甾醇含量的新方法。将（蛋白）脂质体保持在一个封闭的隔室中可最大限度地减少胆固醇转移过程中的材料损失，并有助于有效去除mβCD。我们借助溶剂化显色探针C-Laurdan监测膜中的甾醇浓度，该探针可报告脂质堆积情况。使用Förster共振能量转移，我们表明将胆固醇递送至蛋白脂质体会诱导膜性质传感器的寡聚化，而随后去除胆固醇则显示出完全可逆性。我们提出，在透析装置中通过mβCD介导的胆固醇递送和去除来调节膜压缩性，为研究甾醇和膜压缩性对膜蛋白结构、功能和动力学的影响提供了一种新方法。
