Selective adhesion, lipid exchange and membrane-fusion processes between vesicles of various sizes bearing complementary molecular recognition groups.

Equimolar mixtures of large unilamellar vesicles (LUVs) obtained from mixtures of egg lecithin and lipids containing complementary hydrogen bonding head groups (barbituric acid (BAR) and 2,4,6-triaminopyrimidine (TAP)) were shown to aggregate and fuse. These events have been studied in detail using electron microscopy and dynamic light scattering, and by fluorimetry using membrane or water-soluble fluorescence probes. It was shown that aggregation was followed by two competitive processes: a) lipid mixing leading to redispersion of the vesicles; b) fusion events generating much larger vesicles. In order to better understand the nature of the interaction, the effects of ionic strength and surface concentration of recognition lipids on the aggregation process were investigated by dynamic light scattering. Additionally, it was possible to inhibit the aggregation kinetics through addition of a soluble barbituric acid competitor. The study was extended to giant unilamellar vesicles (GUVs) to investigate the size effect and visualise the phenomena in situ. The interactions between complementary LUVs and GUVs or GUVs and GUVs were studied by optical microscopy using dual fluorescent labelling of both vesicle populations. A selective adhesion of LUVs onto GUVs was observed by electron and optical microscopies, whereas no aggregation took place in case of a GUV/GUV mixture. Furthermore, a fusion assay of GUV and LUV using the difference of size between GUV and LUV and calceine self-quenching showed that no mixing between the aqueous pools occured.

Monomer-oligomer equilibrium of bacteriorhodopsin in reconstituted proteoliposomes. A freeze-fracture electron microscope study.

An improved freeze-fracture electron microscope procedure has been developed and applied to the study of the association of bacteriorhodopsin in large proteoliposomes reconstituted by reverse-phase evaporation with egg lecithin. Due to the improved accuracy and resolution of this procedure, intramembrane particles, the diameter of which (4.5 nm) closely matched that of bacteriorhodopsin monomer, could be observed at high lipid to protein ratios (greater than or equal to 40 w/w). At lower lipid to protein ratios, larger particles (diameter 7.5 nm) progressively appeared, resulting in bimodal particle size distributions up to a lipid to protein ratio of 1, where the large particles were the sole species present. These large particles were interpreted as corresponding to bacteriorhodopsin oligomers. Because of the large size and homogeneity of proteoliposomes, accurate particle density measurements could be performed. These confirmed the occurrence of a lipid to protein ratio-dependent bacteriorhodopsin monomer-oligomer equilibrium and further allowed us to identify the oligomer as a trimer or a tetramer. In complementary experiments, it was found that the bacteriorhodopsin monomer and oligomer had identical visible CD spectra and light-induced proton pumping rates. However, a large increase of the proton passive leak rate of proteoliposomes was found to be associated with oligomer formation. The appearance of these oligomers may be important as the first step in the formation of two-dimensional crystals of bacteriorhodopsin.