Membrane assembly of the 16-kDa proteolipid channel from Nephrops norvegicus studied by relaxation enhancements in spin-label ESR.
The 16-kDa proteolipid from the hepatopancreas of Nephrops norvegicus belongs to the class of channel proteins that includes the proton-translocation subunit of the vacuolar ATPases. The membranous 16-kDa protein from Nephrops was covalently spin-labeled on the unique cysteine Cys54, with a nitroxyl maleimide, or on the functionally essential glutamate Glu140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). The intensities of the saturation transfer ESR spectra are a sensitive indicator of spin-spin interactions that were used to probe the intramembranous structure and assembly of the spin-labeled 16-kDa protein. Spin-lattice relaxation enhancements by aqueous Ni(2+) ions revealed that the spin label on Glu140 is located deeper within the membrane (around C9-C10 of the lipid chains) than is that on Cys54 (located around C5-C6). In double labeling experiments, alleviation of saturation by spin-spin interactions with spin-labeled lipids indicates that spin labels both on Cys54 and on Glu140 are at least partially exposed to the lipid chains. The decrease in saturation transfer ESR intensity observed with increasing spin-labeling level is evidence of oligomeric assembly of the 16-kDa monomers and is consistent with a protein hexamer. These results determine the locations and orientations of transmembrane segments 2 and 4 of the 16-kDa putative 4-helix bundle and put constraints on molecular models for the hexameric assembly in the membrane. In particular, the crucial DCCD-binding site that is essential for proton translocation appears to contact lipid.
Páli
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Max-Planck-Institut für biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, Germany.
Finbow
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Marsh
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D000818:Animals; D002413:Cations, Divalent; D003497:Cyclic N-Oxides; D003545:Cysteine / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide; D004064:Digestive System / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D018698:Glutamic Acid / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism*; D008566:Membranes / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D008121:Nephropidae / Q000737:chemistry / Q000378:metabolism*; D009532:Nickel; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism*; D013113:Spin Labels
D002413:Cations, Divalent; D003497:Cyclic N-Oxides; D007473:Ion Channels; D011510:Proteolipids; D013113:Spin Labels; C087027:proteolipid, Nephrops; D018698:Glutamic Acid; C010638:N-(2,2,6,6-tetramethylpiperidyl-1-oxyl) N'- (cyclohexyl)carbodiimide; D004024:Dicyclohexylcarbodiimide; C024516:N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide; D009532:Nickel; D003545:Cysteine