A collision gradient method to determine the immersiondepth of nitroxides in lipid bilayers: application to spin-labeled mutants ofbacteriorhodopsin.

Abstract

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 ofan a-helix. These results provide a general strategy for determining the immersion depth of nitroxides in bilayers.Site-directed spin labeling has become a powerful tool for determination of membrane protein structures and their disposition with respect to the bilayer (1-3). In previous studies, information on the region where transmembrane helices intersect with the membrane/solution interface has been obtained by analysis of a consecutive series of mutants that traverse the interface (1). If it were possible to determine the vertical distance of a spin-labeled side chain from the plane of the phosphates of the lipid headgroups, analysis of only one or two spin-labeled mutants would be sufficient to determine the position ofa transmembrane domain relative to the membrane.EPR methods for estimation of the depth of immersion of a nitroxide in the membrane have been reported (4-6). These methods are based upon the dipolar interactions of the nitroxide with paramagnetic reagents constrained to the aqueous phase and require knowledge of the spacial distribution of the paramagnetic reagents in solution. This distribution may be readily deduced for a pure bilayer with a nitroxide on a lipid chain, but not for a nitroxide attached to a protein in a bilayer. This is because the protein has an unknown excluded volume for the paramagnetic reagent in the aqueous phase.In this report, we make use of site-directed spin labeling to introduce nitroxides along the entire length of helix D of bacteriorhodopsin (bR). The collision frequencies of the nitroxides with paramagnetic reagents are dependent upon position, and this effect is shown to provide an approach for localization of nitroxides in the membrane interior. MATERIALS AND METHODSEgg yolk phosphatidylcholine (PC) and 1-palmitoyl-2-(ndoxylpalmitoyl) PC spin-labeled isomers with n = 5, 7, 10, 12, and 16 were obtained from Avanti Polar Lipids. Ethylenediamine-N,N'-diacetic acid (EDDA), nickel(II) acetylacetonate (NiAA), and Ni(OH)2 were obtained from Aldrich. bR mutants were prepare...

Aqueous sample in an EPR cavity: sensitivity considerations

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Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

“…Structure and structure-function relationships of macromolecules are areas of intense EPR effort [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Coupled with site-directed spin-labeling (SDSL), EPR is oftentimes used to characterize protein and nucleic acid structures and dynamics, conformational changes, molecule folding, macromolecule complexes, and oligomeric structures [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]17].…”

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Effects of Spin-Labels on Membrane Burial Depth of MARCKS-ED Residues

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