Labeling of subunit b of the ATP synthase from Escherichia coli with a photoreactive phospholipid analogue.
Purified ATP synthase (F1F0) from Escherichia coli K12 was labeled with the hydrophobic photoreactive label 1-palmitoyl 2-(2-azido-4-nitro)benzoyl sn-glycero-3-[3H]phosphocholine in reconstituted proteoliposomes. The F0-subunit b was predominantly labeled. A very low amount of label was detected on the other F0-subunits a and c. The label in subunit b could be traced back by proteolytic digestion to the NH2-terminal fragment 1 to 53 which contains the stretch of hydrophobic amino acid residues 1 to 32. By sequencing the intact protein, the distribution of label among the amino acids in this segment was determined. Cysteine 21 was predominantly labeled. Other labeled amino acids occurred at the NH2-terminal (Asn-2) and at position 26 (tryptophan). Due to the restricted mobility of the label in the lipid bilayer, these residues are suggested to be located in or close to the polar head of the lipid bilayer. These results will be compared with predictions for the arrangement of the polypeptide b derived from the hydrophobicity profile.
D025181:ATP Synthetase Complexes; D000596:Amino Acids / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology*; D010770:Phosphotransferases / Q000378:metabolism*
D000596:Amino Acids; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D010713:Phosphatidylcholines; C037402:1-palmitoyl-2-(2-azido-4-nitro)benzoyl-glycero-3-phosphocholine; D010770:Phosphotransferases; D025181:ATP Synthetase Complexes