title: Peptide building blocks from bacteriorhodopsin: isolation and physicochemical characterization of two individual transmembrane segments.
abstract: 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.
authors.affiliation: Institute of Biochemistry, University of Berne, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Wuethrich
authors.forename: H
authors.initials: H
authors.lastname: Sigrist
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000032:analysis* / Q000378:metabolism; D003488:Cyanogen Bromide / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D008823:Micelles; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism; D012995:Solubility; D012997:Solvents; D014867:Water
chemical_list: D008565:Membrane Proteins; D008823:Micelles; D010446:Peptide Fragments; D012997:Solvents; D014867:Water; D001436:Bacteriorhodopsins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003488:Cyanogen Bromide