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Ligand-induced conformational change in the ferric enterobactin receptor FepA as studied by site-directed spin labeling and time-domain ESR. Biochemistry 1998 Jun 23;37(25):9016-23

Date

06/24/1998

Pubmed ID

9636045

DOI

10.1021/bi980144e

Scopus ID

2-s2.0-0032560604   23 Citations

Abstract

A mutant of the ferric enterobactin receptor, FepA, containing a valine to cysteine (V338C) substitution was made and the purified protein selectively modified with a sulfhydryl-specific nitroxide spin label. In reconstituted liposomes, interaction of the attached spin label with a combination of water-soluble and lipid-soluble relaxation agents indicated that the V338C site was located in the polar headgroup region of the membrane, approximately 1.5-4.5 A above the phosphate groups of the lipids. Binding of the ligand, ferric enterobactin (FeEnt), to the purified spin-labeled protein produced a significant decrease in both the rotational freedom and accessibility of the nitroxide, indicating the formation of new structural contacts between the spin label and either the protein or the bound ligand. Electron spin-echo (ESE) measurements of the nitroxide phase-memory relaxation rate in the presence and absence of bound ligand showed substantial dipolar coupling between the Fe3+ of FeEnt and the spin label and provided an iron-nitroxide distance estimate in the range of 20-30 A. We conclude that the ligand-induced changes in spin label motion and accessibility are due to new tertiary contacts with the protein and not to direct contact with the ligand. These studies suggest that V338C may occupy a hinge region connecting the ligand binding surface loop to the beta-barrel and provide the strongest evidence to date of an in vitro ligand-induced conformational change in FepA.

Author List

Klug CS, Eaton SS, Eaton GR, Feix JB

Authors

Jimmy B. Feix PhD Professor in the Biophysics department at Medical College of Wisconsin
Candice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Bacterial Outer Membrane Proteins
Carrier Proteins
Cysteine
Electron Spin Resonance Spectroscopy
Enterobactin
Ligands
Lipid Bilayers
Liposomes
Mutagenesis, Site-Directed
Protein Conformation
Receptors, Cell Surface
Spin Labels
Valine
jenkins-FCD Prod-400 0f9a74600e4e79798f8fa6f545ea115f3dd948b2