Lipid-protein interactions and assembly of the 16-kDa channel polypeptide from Nephrops norvegicus. Studies with spin-label electron spin resonance spectroscopy and electron microscopy.
The assembly of 16-kDa polypeptide channel units in membranes from the hepatopancreas of Nephrops norvegicus has been studied both by electron microscopy and by the lipid--protein interactions reported with spin-labeled lipids. Membranes prepared by extraction with N-lauroylsarcosine and Triton X-100 have a low lipid/protein ratio (ca. 4-6.5 phospholipids and 1 cholesterol per 16-kDa monomer), and those prepared by alkaline extraction have a higher lipid/protein ratio (ca. 12-16 phospholipids and 3.5-4 cholesterols per 16-kDa monomer). In the membranes extracted with detergents, the protein is assembled in membrane sheets as hexagonally packed hexameric complexes, whereas the alkali-extracted preparations consist of closed vesicles in which the channel complexes are near randomly distributed. The electron spin resonance (ESR) spectra from lipids spin-labeled at the C-14 position of the (sn-2) chain show lower mobility for the membranes extracted with N-lauroylsarcosine than for the alkaline-extracted membranes. At higher temperatures, the ESR spectra reveal a population of lipids whose mobility is restricted by direct interaction with the intramembranous sections of the channel assemblies. The population of protein-associated spin-labeled phosphatidylcholine in the alkali-extracted membranes corresponds to 4-5 phospholipid molecules plus 1 cholesterol molecule per 16-kDa polypeptide monomer.(ABSTRACT TRUNCATED AT 250 WORDS)
Páli
T
T
Abteilung Spektroskopie, Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany.
Finbow
M E
ME
Holzenburg
A
A
Findlay
J B
JB
Marsh
D
D
D000595:Amino Acid Sequence; D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D066298:In Vitro Techniques; D008055:Lipids / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008121:Nephropidae / Q000737:chemistry*; D010179:Pancreas / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D013113:Spin Labels
D008055:Lipids; D011506:Proteins; D011510:Proteolipids; D013113:Spin Labels; C087027:proteolipid, Nephrops