PMID:9335564

Citation	Klug, CS, Su, W and Feix, JB (1997) Mapping of the residues involved in a proposed beta-strand located in the ferric enterobactin receptor FepA using site-directed spin-labeling. Biochemistry 36:13027-33
Abstract	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.
Links	PubMed Online version:10.1021/bi971232m
Keywords	Amino Acid Sequence; Amino Acid Substitution; Bacterial Outer Membrane Proteins/chemistry; Bacterial Outer Membrane Proteins/metabolism; Binding Sites; Carrier Proteins/chemistry; Carrier Proteins/isolation & purification; Carrier Proteins/metabolism; Colicins/pharmacology; Cysteine; Edetic Acid/analogs & derivatives; Electron Spin Resonance Spectroscopy; Enterobactin; Escherichia coli/drug effects; Escherichia coli/growth & development; Escherichia coli/metabolism; Hydroxybutyrates; Lipid Bilayers; Liposomes; Mesylates; Models, Structural; Molecular Sequence Data; Mutagenesis, Site-Directed; Nickel; Oxalates; Oxygen; Pentanones; Protein Structure, Secondary; Receptors, Cell Surface/chemistry; Receptors, Cell Surface/metabolism; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Spin Labels
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