MAPPING OF THE RESIDUES INVOLVED IN A PROPOSED BETA-STRAND LOCATED INTHE FERRIC ENTEROBACTIN RECEPTOR FEPA USING SITE-DIRECTED SPIN-LABELING

Electron paramagnetic resonance (EPR) site-directed spin-labeling (SDS L) has been used to characterize a proposed transmembrane beta-strand of the Escherichia coil ferric enterobactin receptor, FepA. Each of ni ne consecutive residues was mutated to cysteine and subsequently label ed with the sulfhydryl-specific spin-label methanethiosulfonate (MTSL) and the purified protein reconstituted into liposomes. Continuous wav e (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 ethylenediaminediaceta te (NiEDDA), chromium oxalate (CROX), and molecular oxygen. The spin-l abel attached to Q245C, L247C, L249C, A251C, and Y253C had higher coll ision frequencies with molecular oxygen than with polar relaxation age nts, 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 oxyge n, suggesting that these sites are exposed to the aqueous channel. The alternating periodicity observed with the polar relaxation agents, Ni AA and NiEDDA, and in opposite phase with oxygen, is consistent with b eta-sheet structure. Depth measurements, based on the reciprocal conce ntration gradients of NiEDDA and O-2 across the bilayer and calibrated for our system with phosphatidylcholine spin-labels, indicated that L 249C was nearest the center of the bilayer and that Q245C and Y253C we re located just below the bilayer surface in opposite leaflets of the membrane. Thus, we conclude that this approach, through mapping of ind ividual residues, has the capability of defining P-sheet secondary str ucture.