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. From the intrinsic fluorescence measurements as well as from the various quenching experiments, it is evident that the interaction of the signal peptide with negatively charged lipids is stronger than with the zwitterionic PC. The results are consistent with a deeper penetration of the tryptophan-containing part of the signal peptide into the hydrophobic interior of the bilayer when negatively charged lipids are used. It is proposed that the presence of negatively charged lipids in Escherichia coli membranes is essential for a functional interaction of the PhoE signal sequence with these membranes. © 1990, American Chemical Society. All rights reserved.