Reconstitution of bacteriorhodopsin from a mixture of a proteinase V 8 fragment and two synthetic peptides

Abstract

The peptide and retinal mixture of bacteriorhodopsin, composed of two synthetic peptides corresponding to helices F (160–197) and G (202–237) and a proteinase V 8-derived fragment V 1 (1–166), generated the characteristic features of bacteriorhodopsin with absorbance maximum at 550 nm and fluorescence quenching as in two synthetic peptides corresponded to helix A (sequence 7–31) and B (41–65), and a chymotryptic fragment (72–248). The recovery of reconstitution estimated from the absorption and the fluorescence quenching of these mixture was 16–19% and 25–32% of the native purple membrane, respectively, whereas mixtures lacking any one of the peptides exhibited no absorption recovery. Circular dichroism of each peptide fragment showed complete formation of α-helical structure in a membrane-mimetic medium of sodium dodecyl sulfate. These results indicate that the specific interactions or mutual recognitions between α-helices in lipid bilayers are essential for correct bundling of the seven helices and formation of the retinal binding pocket.