Textor Martin, Vargas Carolyn, Keller Sandro
Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany.
Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany.
Methods. 2015 Apr;76:183-193. doi: 10.1016/j.ymeth.2015.01.002. Epub 2015 Jan 10.
Reconstitution from detergent micelles into lipid bilayer membranes is a prerequisite for many in vitro studies on purified membrane proteins. Complexation by cyclodextrins offers an efficient and tightly controllable way of removing detergents for membrane-protein reconstitution, since cyclodextrins sequester detergents at defined stoichiometries and with tuneable affinities. To fully exploit the potential advantages of cyclodextrin for membrane-protein reconstitution, we establish a quantitative model for predicting the supramolecular transition from mixed micelles to vesicles during cyclodextrin-mediated detergent extraction. The model is based on a set of linked equilibria among all pseudophases present in the course of the reconstitution process. Various isothermal titration-calorimetric protocols are used for quantifying a detergent's self-association as well as its colloidal and stoichiometric interactions with lipid and cyclodextrin, respectively. The detergent's critical micellar concentration, the phase boundaries in the lipid/detergent phase diagram, and the dissociation constant of the cyclodextrin/detergent complex thus obtained provide all thermodynamic parameters necessary for a quantitative prediction of the transition from micelles to bilayer membranes during cyclodextrin-driven reconstitution. This is exemplified and validated by stepwise complexation of the detergent lauryldimethylamine N-oxide in mixtures with the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine upon titration with 2-hydroxypropyl-β-cyclodextrin, both in the presence and in the absence of the membrane protein Mistic. The calorimetric approach presented herein quantitatively predicts the onset and completion of the reconstitution process, thus obviating cumbersome trial-and-error efforts and facilitating the rational optimisation of reconstitution protocols, and can be adapted to different cyclodextrin/lipid/detergent combinations.
从去污剂胶束重构到脂质双分子层膜是许多关于纯化膜蛋白的体外研究的前提条件。环糊精络合提供了一种高效且可控性强的去除去污剂以进行膜蛋白重构的方法，因为环糊精能以确定的化学计量比和可调节的亲和力螯合去污剂。为了充分利用环糊精在膜蛋白重构方面的潜在优势，我们建立了一个定量模型，用于预测环糊精介导的去污剂提取过程中从混合胶束到囊泡的超分子转变。该模型基于重构过程中所有假相之间的一组关联平衡。各种等温滴定量热法方案分别用于量化去污剂的自缔合以及它与脂质和环糊精的胶体和化学计量相互作用。由此获得的去污剂的临界胶束浓度、脂质/去污剂相图中的相界以及环糊精/去污剂复合物的解离常数提供了环糊精驱动的重构过程中从胶束到双分子层膜转变的定量预测所需的所有热力学参数。在用2-羟丙基-β-环糊精滴定含有和不含有膜蛋白Mistic的情况下，去污剂月桂基二甲基氧化胺与磷脂1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱混合物的逐步络合对此进行了举例说明和验证。本文提出的量热法方法定量预测了重构过程的开始和完成，从而避免了繁琐的反复试验工作，并有助于合理优化重构方案，并且可以适用于不同的环糊精/脂质/去污剂组合。
