Rémigy H-W, Caujolle-Bert D, Suda K, Schenk A, Chami M, Engel A
M.E. Müller Institute for Microscopy, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland.
FEBS Lett. 2003 Nov 27;555(1):160-9. doi: 10.1016/s0014-5793(03)01105-0.
Efficient reconstitution of membrane proteins for functional analyses can be achieved by dilution of a ternary mixture containing proteins, lipids and detergents. Once the dilution reaches the point where the free detergent concentration would become lower than the critical micellar concentration, detergent is recruited from the bound detergent pool, and association of proteins and lipids is initiated. Here we show that dilution is also suitable for the assembly of two-dimensional crystals. A device has been designed that allows controlled dilution of a protein-lipid-detergent mixture to induce formation of densely packed or crystalline proteoliposomes. Turbidity is used to monitor the progress of reconstitution on-line, while dilution is achieved by computer-controlled addition of buffer solution in sub-microliter steps. This system has mainly been tested with porin OmpF, a typical beta-barrel protein, and aquaporin-1, a typical alpha-helical protein. The results demonstrate that large, highly ordered two-dimensional crystals can be produced by the dilution method.
通过稀释包含蛋白质、脂质和去污剂的三元混合物，可实现用于功能分析的膜蛋白的高效重组。一旦稀释达到游离去污剂浓度将低于临界胶束浓度的程度，去污剂就会从结合的去污剂池中被募集，蛋白质和脂质的缔合随即开始。在此我们表明，稀释也适用于二维晶体的组装。设计了一种装置，可对蛋白质 - 脂质 - 去污剂混合物进行可控稀释，以诱导形成紧密堆积或结晶的蛋白脂质体。通过监测浊度在线跟踪重组过程，同时通过计算机控制以亚微升步长添加缓冲溶液来实现稀释。该系统主要用典型的β - 桶状蛋白孔蛋白OmpF和典型的α - 螺旋蛋白水通道蛋白 - 1进行了测试。结果表明，通过稀释法可产生大的、高度有序的二维晶体。
