DETERGENT-MEDIATED RECONSTITUTION OF MEMBRANE-PROTEINS

The efficiency of reconstitution of the lactose transport protein (Lac S) of Streptococcus thermophilus is markedly higher with Triton X-100 than with other detergents commonly employed to mediate the membrane i nsertion. To rationalize these differences, the lipid/detergent struct ures that are formed in the reconstitution process were studied by cry otransmission electron microscopy. Surprisingly, the two nonionic dete rgents Triton X-100 and n-dodecyl beta-D-maltoside (DDM) affected the liposome structures in a completely different manner. Preformed liposo mes titrated with Triton X-100 maintained their bilayer structure far beyond the onset of solubilization, and transport activity was maximal when LacS was inserted into these structures. With DDM the vesicular structures were already disrupted at the onset of solubilization and t hese membrane sheets were converted into long threadlike micelles at h igher DDM to lipid ratios. Triton X-100 allowed the protein to be reco nstituted with the hydrophilic surface exposed to the outside, whereas LacS was incorporated randomly when DDM was used. These differences i n orientation are readily explained by the different lipid-detergent s tructures formed by Triton X-100 and DDM. The orientation of the recon stituted LacS protein is a critical factor for the activity of the pro tein as the kinetics of translocation is very different for opposite d irections of transport.