Topology of the outer membrane usher PapC determined by site-directed fluorescence labeling.
In contrast to typical membrane proteins that span the lipid bilayer via transmembrane alpha-helices, bacterial outer membrane proteins adopt a beta-barrel architecture composed of antiparallel transmembrane beta-strands. The topology of outer membrane proteins is difficult to predict accurately using computer algorithms, and topology mapping protocols commonly used for alpha-helical membrane proteins do not work for beta-barrel proteins. We present here the topology of the PapC usher, an outer membrane protein required for assembly and secretion of P pili by the chaperone/usher pathway in uropathogenic Escherichia coli. An initial attempt to map PapC topology by insertion of protease cleavage sites was largely unsuccessful due to lack of cleavage at most sites and the requirement to disrupt the outer membrane to identify periplasmic sites. We therefore adapted a site-directed fluorescence labeling technique to permit topology mapping of outer membrane proteins using small molecule probes in intact bacteria. Using this method, we demonstrated that PapC has the potential to encode up to 32 transmembrane beta-strands. Based on experimental evidence, we propose that the usher consists of an N-terminal beta-barrel domain comprised of 26 beta-strands and that a distinct C-terminal domain is not inserted into the membrane but is located instead within the lumen of the N-terminal beta-barrel similar to the plug domains encoded by the outer membrane iron-siderophore uptake proteins.
Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, Stony Brook University, Stony Brook, NY 11794-5120, USA.
Nadine S
NS
Henderson
Stephane Shu Kin
SS
So
Cheryl
C
Martin
Ritwij
R
Kulkarni
David G
DG
Thanassi
D000465:Algorithms; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D006358:Hot Temperature; D008051:Lipid Bilayers / Q000737:chemistry; D046911:Macromolecular Substances / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D018272:Porins / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008565:Membrane Proteins; D018272:Porins; C111243:atpG protein, E coli; D003545:Cysteine