The transmembrane domain of glycophorin A as studied by cross-linking using photoactivatable phospholipids.
Glycophorin A, the major sialoglycoprotein of the human erythrocyte, consists of a NH2-terminal carbohydrate-rich region exposed to the outside, a hydrophobic region which forms a transmembrane bridge, and a COOH-terminal hydrophilic region extending into the cytoplasm. With the aim of further defining the membrane-embedded region, the protein has been reconstituted into vesicles formed from dimyristoylphosphatidylcholine and phospholipids containing photosensitive carbene precursors. The photosensitive groups were incorporated either at the omega-position of sn-2 fatty acyl chain or the polar head group of lecithins. Following photolysis, covalent cross-linking (1-2%) of the photoactivatable phospholipids to the protein was demonstrated. Degradation and sequence analysis showed that in the case of phospholipids containing photoactivatable groups in the fatty acyl chains most of the covalent cross-linking involved the carboxyl group of Glu-70. Therefore, the latter residue must be within the bilayer. This conclusion was supported by the reaction of the membrane-permeant [14C] dicyclohexylcarbodiimide with glycophorin reconstituted into vesicles. The same residue was labeled. Photolysis of glycophorin vesicles containing phospholipids with photolabels in the polar head group gave products in which the cross-links were present in peptide fragments (residues 62-81 and 82-96). These results define the probable boundaries of the membrane-embedded segment of glycophorin A. Corresponding experiments with erythrocyte ghosts gave similar results.
D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D004024:Dicyclohexylcarbodiimide; D004134:Dimyristoylphosphatidylcholine; D004910:Erythrocyte Membrane; D006021:Glycophorins; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers; D010446:Peptide Fragments / Q000032:analysis; D010713:Phosphatidylcholines; D010743:Phospholipids; D010782:Photolysis; D011485:Protein Binding; D012795:Sialoglycoproteins
D003432:Cross-Linking Reagents; D006021:Glycophorins; D008051:Lipid Bilayers; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010743:Phospholipids; D012795:Sialoglycoproteins; D004024:Dicyclohexylcarbodiimide; D003488:Cyanogen Bromide; D004134:Dimyristoylphosphatidylcholine