Christian Altenbach1, David A. Greenhalgh1, H G Khorana1, Wayne L. Hubbell1
1Jules Stein Eye Institute, Los Angeles, CA.
Tóm tắt
Ten mutants of bacteriorhodopsin, each containing
a single cysteine residue regularly spaced along helix D and facing the lipid
bilayer, were derivatized with a nitroxide spin label. Collision rates of the
nitroxide with apolar oxygen increased with distance from the membrane/solution
interface. Collision rates with polar metal ion complexes decreased over the
same distance. Although the collision rates depend on steric constraints imposed
by the local protein structure and on the depth in the membrane, the ratio of
the collision rate of oxygen to those of a polar metal ion complex is
independent of structural features of the protein. The logarithm of the ratio is
a linear function of depth within the membrane. Calibration of this ratio
parameter with spin-labeled phospholipids allows localization of the individual
nitroxides, and hence the bacteriorhodopsin molecule, relative to the plane of
the phosphate groups of the bilayer. The spacing between residues is consistent
with the pitch of an alpha-helix. These results provide a general strategy for
determining the immersion depth of nitroxides in bilayers.
