Selective detection of the rotational dynamics of the protein-associated lipid hydrocarbon chains in sarcoplasmic reticulum membranes.
We have developed a saturation transfer EPR (ST-EPR) method to measure selectively the rotational dynamics of those lipids that are motionally restricted by integral membrane proteins and have applied this methodology to measure lipid-protein interactions in native sarcoplasmic reticulum (SR) membranes. This analysis involves the measurement of spectral saturation using a series of six stearic acid spin labels that are labeled with a nitroxide at different carbon atom positions. A large amount of spectral saturation is observed for spin labels in native SR membranes, but not for spin labels in dispersions of extracted SR lipids, implying that the motional properties of those lipids interacting with the Ca-ATPase, i.e., the boundary or annular lipid, can be directly measured without the need for spectral subtraction procedures. A comparison of the motional properties of the restricted lipid, measured by ST-EPR, with those measured by digital subtraction of conventional EPR spectra qualitatively agree, for in both cases the Ca-ATPase restricts the rotational mobility of a population of lipids, whose rotational mobility increases as the nitroxide is positioned toward the center of the bilayer. However, the ability of ST-EPR to directly measure the motionally restricted lipid in a model-independent means provides the greater precision necessary to measure small changes in the rotational dynamics of the lipid at the protein-lipid interface, providing a valuable tool in clarifying the relationship between the physical nature of the protein-lipid interface and membrane function.
Squier
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Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Thomas
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D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D008051:Lipid Bilayers; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013113:Spin Labels; D013816:Thermodynamics
D008051:Lipid Bilayers; D008563:Membrane Lipids; D008565:Membrane Proteins; D013113:Spin Labels; D000252:Calcium-Transporting ATPases