A divalent-ion binding site on the 16-kDa proton channel from Nephrops norvegicus--revealed by EPR spectroscopy.
As purified from the hepatopancreas of Nephrops norvegicus, the 16-kDa proton channel proteolipid is found to contain an endogenous divalent ion binding site that is occupied by Cu2+. The EPR spectrum has g-values and hyperfine splittings that are characteristic of type 2 Cu2+. The copper may be removed by extensive washing with EDTA. Titration with Ni2+ then induces spin-spin interactions with nitroxyl spin labels that are attached either to the unique Cys54, or to fatty acids intercalated in the membrane. Paramagnetic relaxation enhancement by the fast-relaxing Ni2+ is used to characterise the binding and to estimate distances from the dipolar interactions. The Ni2+-binding site on the protein is situated around 14-18 A from the spin label on Cys54, and is at a similar distance from a lipid chain spin-labelled on the 5 C-atom, but is more remote from the C-9 and C-14 positions of the lipid chains.
Max-Planck-Institut für biophysikalische Chemie, Abteilung Spektroskopie, 37070 Göttingen, Germany.
Tibor
T
Páli
Malcolm E
ME
Finbow
Derek
D
Marsh
D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism*; D008970:Molecular Weight; D008121:Nephropidae / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism
D002413:Cations, Divalent; D007473:Ion Channels; D011510:Proteolipids; D003300:Copper; D009532:Nickel; D025262:Vacuolar Proton-Translocating ATPases