A ruler for determining the position of proteins in membranes.
Both the oxygen diffusion rate and the oxygen solubility vary with depth into the interior of biological membranes. The product of these two gradients generates a single gradient, a permeability gradient, which is a smooth continuous function of the distance from the center of the membrane. Using electron paramagnetic resonance and the spin-probe method, the relaxation gradient of oxygen, which is directly proportional to the permeability gradient, is the quantity that can be directly measured in membranes under physiological conditions. The gradient obtained provides a calibrated ruler for determining the membrane depth of residues either from loop regions of membrane-binding proteins or from the membrane-exposed residues of transmembrane proteins. We have determined the relaxation gradient of oxygen in zwitterionic and anionic phospholipid membranes by attaching a single nitroxide probe to a transmembrane alpha-helical polypeptide at specific residues. The peptide ruler was used to determine the depth of penetration of the calcium-binding loops of the C2 domain of cytosolic phospholipase A(2). The positions of selected residues of this membrane-binding protein that penetrate into the membrane, determined using this ruler, compared favorably with previous determinations using more complex methods. The relaxation gradient constrains the possible values of the membrane-dependent oxygen concentration and the oxygen diffusion gradients. The average oxygen diffusion coefficient is estimated to be at least 2-fold smaller in the membrane than that in water.
Nielsen
Robert D
RD
Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195, USA.
Che
Kepeng
K
Gelb
Michael H
MH
Robinson
Bruce H
BH
D003600:Cytosol / Q000201:enzymology; D004058:Diffusion; D004578:Electron Spin Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D019906:Nuclear Magnetic Resonance, Biomolecular; D010100:Oxygen / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization; D013113:Spin Labels
D008565:Membrane Proteins; D010455:Peptides; D010743:Phospholipids; D013113:Spin Labels; D010741:Phospholipases A; D010100:Oxygen