Triton X −100 and TM Helices Increase Ordered Domain (lipid Raft) Size

Abstract

It has been postulated that the formation of co-existing liquid ordered (Lo) domains rich in sphingolipids and cholesterol and liquid disordered (Ld) domains rich in unsaturated lipids, has an important role in cell membrane structure and function. Detergent TX-100 favorably partitions into and dissolves the Ld phase. It is used to isolate insoluble Lo-like membranes known as detergent resistant membrane (DRM) from cells. The relationship between DRM and pre-existing Lo domains in cells is not clear. We carried out experiments to find out how TX-100, and TM helices (which are cell membranes components), change membrane properties by investigating their effect on liposomes composed of brain sphingomyelin/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol. As measured by anisotropy and tempo quenching, methods that detect local environment at the nearest neighbor level, neither TX-100 nor TM helices affected the thermal stability of membrane order. In contrast, FRET, which detects proximity at longer length scales, detected that ordered domains disappeared at a lower temperature than that estimated from anisotropy or quenching, both with and without TX-100 and TM helices. FRET detected domains at higher temperatures in the presence of TX-100 or TM peptides than in their absence. The amount of ordered domains and their thermal stability appeared to increase as the interaction distance (Ro) of the FRET pair decreased. These differences are most easily explained by a difference in the size-sensitivity of the detection techniques, such that FRET is unable to detect Lo domains smaller than Ro in size, and by the conclusion that TX-100 increases ordered domain size. However, in presence of TM peptides, this increase in domain size is less evident, as the TM peptide by itself already increases domain size. Thus, in natural membranes the effect of TX-100 upon domain formation may not be significant.