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)