Lipid-protein surface films generated from membrane vesicles: selfassembly, composition, and film structure

Lipid-protein films at the air-water interface were generated from a variety of native vesicles and from vesicles derived from lipid extracts. A technique is described which is particularly suitable for the generation of films from small amounts of material at high yield and velocity. In all instances, 10 microliters vesicle suspensions containing 25 micrograms protein yield at least 50 cm2 film area at a constant surface pressure of 12 mN/m within minutes. Upon formation, surface films are separated from vesicles by use of shear forces. Complete separation is demonstrated by electron microscopy and surface pressure-area diagrams. The latter confirms previous conclusions that surface films generated from lipid vesicles are organized as a monolayer. Analysis of lipid-protein surface layers reveals that their lipid to protein ratios match those of the vesicles used, within a factor of two, irrespective of whether films are generated at high or low surface pressure. Surface denaturation of membrane proteins is shown to be effectively prevented when the film is generated and held at high surface pressure (greater than or equal to 15 mN/m). Upon surface pressure jumps from high to low values, denaturation kinetics revealed activation areas of 1.5 (+/- 0.2) nm2.