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Mapping of the residues involved in a proposed beta-strand located in the ferric enterobactin receptor FepA using site-directed spin-labeling. Biochemistry 1997 Oct 21;36(42):13027-33



Pubmed ID




Scopus ID

2-s2.0-0030693909   51 Citations


Electron paramagnetic resonance (EPR) site-directed spin-labeling (SDSL) has been used to characterize a proposed transmembrane beta-strand of the Escherichia coli ferric enterobactin receptor, FepA. Each of nine consecutive residues was mutated to cysteine and subsequently labeled with the sulfhydryl-specific spin-label methanethiosulfonate (MTSL) and the purified protein reconstituted into liposomes. Continuous wave (CW) power saturation methods were used to determine exposure of the nitroxide side chains to a series of paramagnetic relaxation agents, including nickel acetylacetonate (NiAA), nickel ethylenediaminediacetate (NiEDDA), chromium oxalate (CROX), and molecular oxygen. The spin-label attached to Q245C, L247C, L249C, A251C, and Y253C had higher collision frequencies with molecular oxygen than with polar relaxation agents, indicating that these sites are exposed to the hydrophobic phase of the lipid bilayer. MTSL bound to residues S246C, E248C, E250C, and G252C had higher collision rates with the polar agents than with oxygen, suggesting that these sites are exposed to the aqueous channel. The alternating periodicity observed with the polar relaxation agents, NiAA and NiEDDA, and in opposite phase with oxygen, is consistent with beta-sheet structure. Depth measurements, based on the reciprocal concentration gradients of NiEDDA and O2 across the bilayer and calibrated for our system with phosphatidylcholine spin-labels, indicated that L249C was nearest the center of the bilayer and that Q245C and Y253C were located just below the bilayer surface in opposite leaflets of the membrane. Thus, we conclude that this approach, through mapping of individual residues, has the capability of defining beta-sheet secondary structure.

Author List

Klug CS, Su W, Feix JB


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

Amino Acid Sequence
Amino Acid Substitution
Bacterial Outer Membrane Proteins
Binding Sites
Carrier Proteins
Edetic Acid
Electron Spin Resonance Spectroscopy
Escherichia coli
Lipid Bilayers
Models, Structural
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Structure, Secondary
Receptors, Cell Surface
Recombinant Proteins
Spin Labels
jenkins-FCD Prod-468 69a93cef3257f26b866d455c1d2b2d0f28382f14