Fluorescence studies on the interaction of a synthetic signal peptide and its analog with liposomes.
The N-terminal signal sequence of glucitol permease of Escherichia coli (Gut22: MIETITPGAVWFIGLFQKGGEC) and its analog (Gut22Ana: MIETITHGAEWFIGLFQKGGEC) were synthesized. The analog had a Pro residue substituted for the His at the 7th position of Gut22 and a Val residue substituted for the Glu at the 10th position. Previous studies indicated that due to its structural rigidity, the interaction of Gut22Ana with lipid bilayer was much weaker than that of Gut22 (Wang, Q.D., Cui, D.F. and Lin, Q.S. (1996) Science in China (Series C) 39, 395-405). To further probe the location of the tryptophan residues of the peptides in lipid bilayer, the membrane penetration depth of the tryptophan residue of Gut22 was measured using spin-labeled phospholipids, and fluorescence quenching of the peptides by iodide and acrylamide in the presence and absence of phosphatidylserine/phosphatidylcholine liposomes were also studied. Fluorescent labeling of the peptides enabled the study of their association with membrane by fluorospectrophotometry. In the presence of liposomes, the peptides were protected from reaction with chymotrypsin, indicating that the peptide incorporated into the membrane. However, dithionite, which acts external to the membrane, reacted with the peptide, showing that the peptides did not translocate across lipid bilayer spontaneously.
Wang
Q
Q
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Academia Sinica, China.
Cui
D
D
Lin
Q
Q
D020106:Acrylamide; D000178:Acrylamides; D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000494:pharmacology; D004227:Dithionite / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D008081:Liposomes / Q000737:chemistry*; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D021382:Protein Sorting Signals / Q000138:chemical synthesis / Q000737:chemistry*; D012974:Sodium Iodide; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D000178:Acrylamides; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D021382:Protein Sorting Signals; D004227:Dithionite; D020106:Acrylamide; D014364:Tryptophan; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C058558:phosphoenolpyruvate glucitol phosphotransferase; D002918:Chymotrypsin; D012974:Sodium Iodide