Peptide building blocks from bacteriorhodopsin: isolation and physicochemical characterization of two individual transmembrane segments.

For protein engineering purposes, transmembrane segments of the structurally stable protein bacteriorhodopsin have been isolated and chemically characterized. Bacteriorhodopsin was cleaved by protease V8 from Staphylococcus aureus to two fragments, V-1 and V-2. The V-2 fragment was separated by gel filtration in organic solvents and purified by reversed-phase FPLC. The fragment has been identified as the C-terminal, partially truncated double-loop of bacteriorhodopsin, including amino acids Val-167-Glu-232/4. Cleavage of V-2 by cyanogen bromide at the single Met-209 yielded two subfragments, which were purified to homogeneity by FPLC procedures. The N-terminal subfragment psi, consisted of a single transmembrane segment (helix F) of bacteriorhodopsin (Val-167-Met(Hse)-209). The C-terminal amphipathic subfragment omega, (Val-210-Glu-232/4) was identified as part of the C-terminal seventh helix of bacteriorhodopsin. Secondary structures of V-2, psi, and omega were investigated in organic solvents and micellar solutions. Native helical structures were partially retained in the solvent systems mentioned.