Oligomeric States and Siderophore Binding of the Ligand-Gated FhuA Protein that Forms Channels Across Escherichia Coli Outer Membranes

Authors


J. P. Rosenbusch, Biozentrum, University of Basel Klingelberstr. 70, CH-4056 Basel, Switzerland
Phone:+41 61 267 2110.
Fax: 141 61 267 2118.
E-mail:rosenbusch@ubaclu.unibas.ch

Abstract

The channel-forming FhuA protein, which translocates ferrichrome across Escherichia coli outer membranes, binds 1 mol ligand/mol monomer in detergent solution. The protein is homogenous and migrates as a single band with a mobility corresponding to 77 kDa in SDS/PAGE electrophoresis. Analytical ultracentrifugation revealed a monodisperse species (S20,w= 3.8 S) with a mass of 77 800 ± 3200 Da. The properties of ligand binding, determined by two independent methods, revealed one binding site/ monomer, but are complicated by a pronounced convexity of the Scatchard plot and a Hill coefficient calculated to be 2.5. This strongly suggests that oligomeric species are present. Cross-linking agents revealed the existence of possibly transient, mostly dimeric and trimeric species. The difference between the FhuA protein in detergent solution and in its native membrane environment may be related to the removal of lateral pressure that exists in situ.

Abbreviations.
DecGlc2

decyl maltoside

LPS

lipopolysaccharide

OcGlc

n-octyl β-d-glucopyranoside

HIO4/Schiff

periodic acid-Schiff staining

Ancillary