Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer

Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the i ntegral membrane protein bacteriorhodopsin. Each system consists of lipid v esicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon ch ains: either L-alpha-1,2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1 ,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamin e lipid increases the desire of each monolayer leaflet in the bilayer to cu rve toward water. This increases the torque tension of such monolayers, whe n they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we hav e used excimer fluorescence from pyrene-labeled phosphatidylcholine Lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regenerati on yield decreases as the mole fraction of the corresponding phosphatidylet hanolamine component is increased. The decrease in yield correlates with th e increase in lateral pressure which the Lipid chains exert on the refoldin g protein. We suggest that the increase in lipid chain pressure either hind ers insertion of the denatured state of bacterioopsin into the bilayer or s lows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.