School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA.
Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA, USA.
Methods Mol Biol. 2021;2302:21-35. doi: 10.1007/978-1-0716-1394-8_2.
Reconstitution of detergent-solubilized membrane proteins into phospholipid bilayers allows for functional and structural studies under close-to-native conditions that greatly support protein stability and function. Here we outline the detailed steps for membrane protein reconstitution to result in proteoliposomes and nanodiscs. Reconstitution can be achieved via a number of different strategies. The protocols for preparation of proteoliposomes use detergent removal via dialysis or via nonpolar polystyrene beads, or a mixture of the two methods. In this chapter, the protocols for nanodiscs apply polystyrene beads only. Proteoliposome preparation methods allow for substantial control of the lipid-to-protein ratio, from minimal amounts of phospholipid to high concentrations, type of phospholipid, and mixtures of phospholipids. In addition, dialysis affords a fairly large degree of control and variation of parameters such as rate of reconstitution, temperature, buffer conditions, and proteoliposome size. For the nanodisc approach, which is highly advantageous for ensuring equal access to both membrane sides of the protein as well as fast reconstitution of only a single membrane protein into a well-defined bilayer environment in each nanodisc, the protocols outline how a number of these parameters are more restricted in comparison to the proteoliposome protocols.
去污剂溶解的膜蛋白重构成磷脂双层，可以在接近天然的条件下进行功能和结构研究，极大地支持蛋白质的稳定性和功能。在这里，我们概述了将膜蛋白重构成脂质体和纳米盘的详细步骤。可以通过多种不同的策略来实现重构成。制备脂质体的方案通过透析或非极性聚苯乙烯珠去除去污剂，或这两种方法的混合物来实现。在本章中，纳米盘的方案仅应用聚苯乙烯珠。脂质体制备方法允许对脂质与蛋白质的比例进行大量控制，从最少量的磷脂到高浓度、磷脂类型和磷脂混合物。此外，透析提供了相当大的控制和参数变化的自由度，如重构成速率、温度、缓冲条件和脂质体大小。对于纳米盘方法，由于它非常有利于确保蛋白质的两个膜面都能平等进入，以及仅将单个膜蛋白快速重构成每个纳米盘中的定义明确的双层环境，因此方案概述了与脂质体方案相比，这些参数中的许多参数受到更多限制。
