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 belon gs to the class of channel proteins that includes the proton-translocation subunit of the vacuolar ATPases. The membranous 16-kDa protein from Nephrop s was covalently spin-labeled on the unique cysteine Cys54, with a nitroxyl maleimide, or on the functionally essential glutamate Glu140, with a nitro xyl analogue of dicyclohexylcarbodiimide (DCCD). The intensities of the sat uration transfer ESR spectra are a sensitive indicator of spin-spin interac tions that were used to probe the intramembranous structure and assembly of the spin-labeled 16-kDa protein. Spin-lattice relaxation enhancements by a queous Ni2+ ions revealed that the spin label on Glu140 is located deeper w ithin the membrane (around C9-C10 of the lipid chains) than is that on Cys5 4 (located around C5-C6). In double labeling experiments, alleviation of sa turation by spin-spin interactions with spin-labeled lipids indicates that spin labels both on Cys54 and on Glu140 are at least partially exposed to t he 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 mod els for the hexameric assembly in the membrane. In particular, the crucial DCCD-binding site that is essential for proton translocation appears to con tact lipid.