A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin.

摘要

Ten mutants of bacteriorhodopsin, each containinga single cysteine residue regularly spaced along helix D and facing the lipidbilayer, were derivatized with a nitroxide spin label. Collision rates of thenitroxide with apolar oxygen increased with distance from the membrane/solutioninterface. Collision rates with polar metal ion complexes decreased over thesame distance. Although the collision rates depend on steric constraints imposedby the local protein structure and on the depth in the membrane, the ratio ofthe collision rate of oxygen to those of a polar metal ion complex isindependent of structural features of the protein. The logarithm of the ratio isa linear function of depth within the membrane. Calibration of this ratioparameter with spin-labeled phospholipids allows localization of the individualnitroxides, and hence the bacteriorhodopsin molecule, relative to the plane ofthe phosphate groups of the bilayer. The spacing between residues is consistentwith the pitch of an alpha-helix. These results provide a general strategy fordetermining the immersion depth of nitroxides in bilayers.