Cs. Klug et al., MAPPING OF THE RESIDUES INVOLVED IN A PROPOSED BETA-STRAND LOCATED INTHE FERRIC ENTEROBACTIN RECEPTOR FEPA USING SITE-DIRECTED SPIN-LABELING, Biochemistry, 36(42), 1997, pp. 13027-13033
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.