Molecular Biophysics, University of Kaiserslautern , Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany.
Anal Chem. 2014 Jan 7;86(1):920-7. doi: 10.1021/ac403723t. Epub 2013 Dec 24.
Phase diagrams offer a wealth of thermodynamic information on aqueous mixtures of bilayer-forming lipids and micelle-forming detergents, providing a straightforward means of monitoring and adjusting the supramolecular state of such systems. However, equilibrium phase diagrams are of very limited use for the reconstitution of membrane proteins because of the occurrence of irreversible, unproductive processes such as aggregation and precipitation that compete with productive reconstitution. Here, we exemplify this by dissecting the effects of the K(+) channel KcsA on the process of bilayer self-assembly in a mixture of Escherichia coli polar lipid extract and the nonionic detergent octyl-β-d-glucopyranoside. Even at starting concentrations in the low micromolar range, KcsA has a tremendous impact on the supramolecular organization of the system, shifting the critical lipid/detergent ratios at the onset and completion of vesicle formation by more than 2-fold. Thus, equilibrium phase diagrams obtained for protein-free lipid/detergent mixtures would be misleading when used to guide the reconstitution process. To address this issue, we demonstrate that, even under such nonequilibrium conditions, high-sensitivity isothermal titration calorimetry can be exploited to monitor the progress of membrane-protein reconstitution in real time, in a noninvasive manner, and at high resolution to yield functional proteoliposomes with a narrow size distribution for further downstream applications.
相图为双层形成脂质和胶束形成去污剂的水相混合物提供了丰富的热力学信息，为监测和调整此类系统的超分子状态提供了一种直接的方法。然而，由于不可逆的、非生产性的过程（如聚集和沉淀）与生产性再组装竞争，平衡相图对于膜蛋白的再组装非常有限。在这里，我们通过剖析钾通道 KcsA 对大肠杆菌极性脂质提取物和非离子去污剂辛基-β-d-吡喃葡萄糖苷混合物中双层自组装过程的影响来说明这一点。即使在起始浓度低至微摩尔范围内，KcsA 对系统的超分子组织也有巨大影响，使囊泡形成开始和完成时的临界脂质/去污剂比增加了 2 倍以上。因此，用于指导再组装过程的不含蛋白质的脂质/去污剂混合物的平衡相图将产生误导。为了解决这个问题，我们证明了，即使在这种非平衡条件下，高灵敏度等温滴定量热法也可以用于实时、非侵入性和高分辨率地监测膜蛋白再组装的进展，从而生成具有窄粒径分布的功能性脂质体，用于进一步的下游应用。
