title: Topography of surface-exposed amino acids in the membrane protein bacteriorhodopsin determined by proteolysis and micro-sequencing.
abstract: The topography of membrane-surface-exposed amino acids in the light-driven proton pump bacteriorhodopsin (BR) was studied. By limited proteolysis of purple membrane with papain or proteinase K, domains were cleaved, separated by SDS-PAGE, and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Fragments transferred were sequenced in a gas-phase sequencer. Papain cleavage sites at Gly-65, Gly-72, and Gly-231, previously only deduced from the apparent molecular weight of the digestion fragments, could be confirmed by N-terminal micro-sequencing. By proteinase K, cleavage occurred at Gln-3, Phe-71, Gly-72, Tyr-131, Tyr-133, and Ser-226, i.e., in regions previously suggested to be surface-exposed. Additionally, proteinase-K cleavage sites at Thr-121 and Leu-127 were identified, which are sites predicted to be in the alpha-helical membrane-spanning segment D. Our results, especially that the amino acids Gly-122 to Tyr-133 are protruding into the aqueous environment, place new constraints on the amino-acid folding of BR across the purple membrane. The validity of theoretical prediction methods of the secondary structure and polypeptide folding for membrane proteins is challenged. The results on BR show that micro-sequencing of peptides separated by SDS-PAGE and blotted to PVDF can be successfully applied to the study of membrane proteins.
authors.affiliation: Department of Physics, Freie Universität Berlin, F.R.G.
authors.forename: S
authors.initials: S
authors.lastname: Fimmel
authors.forename: T
authors.initials: T
authors.lastname: Choli
authors.forename: N A
authors.initials: NA
authors.lastname: Dencher
authors.forename: G
authors.initials: G
authors.lastname: Büldt
authors.forename: B
authors.initials: B
authors.lastname: Wittmann-Liebold
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids; D001436:Bacteriorhodopsins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011487:Protein Conformation; D014961:X-Ray Diffraction
chemical_list: D000596:Amino Acids; D001436:Bacteriorhodopsins