Tryptophan fluorescence study on the interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with model membranes.
The interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with different phospholipid vesicles was investigated by fluorescence techniques, using a synthetic mutant signal peptide in which valine at position -8 in the hydrophobic sequence was replaced by tryptophan. First it was established that this mutation in the signal sequence of prePhoE does not affect in vivo and in vitro translocation efficiency and that the biophysical properties of the synthetic mutant signal peptide are similar to those of the wild-type signal peptide. Next, fluorescence experiments were performed which showed an increase in quantum yield and a blue shift of the emission wavelength maximum upon interaction of the signal peptide with lipid vesicles, indicating that the tryptophan moiety enters a more hydrophobic environment. These changes in intrinsic fluorescence were found to be more pronounced in the presence of phosphatidylglycerol (PG) or cardiolipin (CL) than with phosphatidylcholine (PC). In addition, quenching experiments demonstrated a shielding of the tryptophan fluorescence from quenching by the aqueous quenchers iodide and acrylamide upon interaction of the signal peptide with lipid vesicles, a shielding in the case of acrylamide that was more pronounced in the presence of negatively charged lipids. Finally it was found that acyl chain brominated lipids incorporated into phospholipid bilayers were able to quench the tryptophan fluorescence of the signal peptide, with the quenching efficiency in CL vesicles being much higher than in PC vesicles. The results clearly demonstrate that the PhoE signal peptide interacts strongly with different lipid vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Killian
J A
JA
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
Keller
R C
RC
Struyvé
M
M
de Kroon
A I
AI
Tommassen
J
J
de Kruijff
B
B
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D002308:Cardiolipins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D008051:Lipid Bilayers / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D018272:Porins; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan
D001425:Bacterial Outer Membrane Proteins; D002308:Cardiolipins; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D018272:Porins; D021382:Protein Sorting Signals; D014364:Tryptophan