Importance of detergent and phospholipid in the crystallization of the human erythrocyte anion-exchanger membrane domain.
Three-dimensional crystals were obtained for the membrane domain of the human erythrocyte anion exchanger (AE1, Band 3). Protein homogeneity and stability and the delicate balance between the detergent used and the amount of phospholipids copurifying are critical to the formation of three-dimensional crystals of the AE1 membrane domain. While deglycosylation improved the protein homogeneity, its stability was significantly increased by inhibitor binding. Size-exclusion chromatography showed that the protein was monodisperse in detergents with acyl chains of 10-12 carbons over a pH range of 5.5-10.0. This pH range and the detergents that retained the protein's monodispersity were used for crystallization screening. Crystals were obtained with the protein purified in C(12)E(8), dodecylmaltoside, decylthiomaltoside, and cyclohexyl-hexylmaltoside. Five to 13 lipid molecules per protein were required for the protein crystal formation. Those crystals grown in dodecylmaltoside diffracted X-rays to 14 A. With these factors taken into consideration, ways to further improve the crystal quality are suggested.
Lemieux
M Joanne
MJ
Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York 10016, USA.
Reithmeier
Reinhart A F
RA
Wang
Da-Neng
DN
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000032:analysis*; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001457:Anion Exchange Protein 1, Erythrocyte; D003902:Detergents; D010743:Phospholipids; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid