Accessibility of lysyl residues of Escherichia coli B/r porin (OmpF) to covalent labeling reagents of different sizes. An approach for a three-dimensional structure of a channel-forming protein.
The three-dimensional structure of Escherichia coli B/r porin (OmpF) was studied by chemical modification using activated sugars of different size. Galactose and galactosides of different penetration properties through the porin channel were oxidized by galactose oxidase, and the 6-aldehydes formed were linked to amino groups in porin by reduction with NaBH3CN. Tryptic fragments of modified and unmodified porin were separated by reversed-phase high pressure liquid chromatography and identified by amino acid and amino-terminal analysis from the known primary structure of OmpF. Modification of purified native porin trimers in beta-octylglucoside revealed three classes of amino groups: (i) those not modified by any sugars; (ii) those modified only by small sugars that diffuse rapidly through the pore, such as galactose or melibiose; and (iii) those modified by either small or large sugars, the latter including pore-impermeant sugars such as stachyose. The results suggest that the three classes of amino groups correspond, respectively, to groups buried in the trimeric molecule, those in the interior of the pore and those exposed on the surface of porin. In addition modification experiments performed on whole cells suggested that all the reactive groups modified by the pore-impermeant sugars (class iii) are located on the surface of porin exposed on the outside of the outer membrane.
Schlaeppi
J M
JM
Ichihara
S
S
Nikaido
H
H
D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis*; D008239:Lysine / Q000032:analysis*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010539:Permeability; D018272:Porins; D014357:Trypsin / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D002241:Carbohydrates; D046911:Macromolecular Substances; D010446:Peptide Fragments; D018272:Porins; D014357:Trypsin; D008239:Lysine