In situ characterization of functional purple membrane monolayers at the air-water interface

The purple membrane (PM) of Halobacterium salinarum contains a single type of protein, bacterio-rhodopsin (bR), which is a member of the seven alpha-helices transmembrane protein family. This protein is a photoactive proton pump, translocating one proton from the cytoplasmic to the extracellular side of the PM per photon absorbed. bR is found in trimers in PM, where they are assembled in a two-dimensional hexagonal lattice. We show herein that stable and functional films can be built in monolayers at the air-water interface by spreading aqueous suspensions of purified and native PM patches. In situ spectroscopic measurements at the air-water interface indicate that bR remains photoactive in this environment. Physical parameters of these PM films, such as protein molecular area, irreversible in-plane aggregation, z-axis orientation, film thickness, and surface roughness, were determined from surface pressure and surface potential-area isotherms, fluorescence spectroscopy, and X-ray reflectivity at the air-water interface. We find that PM do form organized monolayers of membranes, with an optimal packing density at a surface pressure of approximately 20 mN/m, although no preferential vectorial alignment, with respect to the plane normal to the membrane, can be detected from fluorescence quenching experiments.