The Phase Property of Membrane Phospholipids Is Affected by the Functionality of Signal Peptides from the Escherichia coli Ribose-binding Protein

Abstract

We examined the effects of synthetic signal peptides from the wild-type, export-defective mutant and its revertant species of ribose-binding protein on the phase properties of lipid bilayers. The lateral segregation of phosphatidylglycerol (PG) in the lipid bilayer was detected through quenching between NBD-PGs upon the reconstitution of signal peptide into the liposome made with the Escherichia coli inner membrane composition. The tendency of lipid segregation was highly dependent on the export competency of signal peptides in vivo, with a decreasing order of wild-type, revertant, and mutant species. The colocalizations of pyrene-PG with BODIPY-PG were also induced by the signal peptides, confirming the phase separation of the acidic phospholipid. The wildtype and revertant signal peptides predominantly formed ␣-helical conformations with the presence of acidic phospholipid as determined by circular dichroism spectroscopy. In addition, they restricted the motion of lipid acyl chains as monitored by fluorescence anisotropy of DPH, suggesting a deep penetration of signal peptide into the lipid bilayer. However, the ␣-helical content of mutant signal peptide was only about half that of the wild-type or revertant peptide with a significantly smaller degree of penetration into the bilayer. An association of the defective signal peptides into the membrane was affected by salt extraction, whereas the functional ones were not. The aforementioned results indicate that the functionality of signal peptide is accomplished through its topologies in the membrane and also by its ability to induce lateral segregation of acidic phospholipid. We propose that the clustering of acidic phospholipid by the functional signal peptide is responsible for the formation of non-bilayer membrane structure, thereby promoting an efficient translocation of secretory proteins.

Decoding the Membrane Activity of the Cyclotide Kalata B1

“…The ability of kB1 to bind to bacteria-like membranes was further investigated with model membranes of compositions similar to the E. coli outer membrane (35) or inner membrane (36,37). The outer membrane contains LPS in the outer layer and phospholipids in the inner layer, whereas the inner membrane comprises a phospholipid bilayer composed mainly of PE and PG phospholipids (37).…”

The Sec System: Protein Export in Escherichia coli

“…The penetration of the peptide into the hydrocarbon lipid tails induces lateral segregation resulting in clustering of PG (364). The hydrophobic region of signals ideally comprises 10 leucine or isoleucine residues (365), which is too short to span the bilayer as an α-helix.…”

The role of lipids in membrane insertion and translocation of bacterial proteins

“…Moreover, synthetic signal peptides were shown to induce clustering of PG, as measured after incorporation into liposomes containing fluorescent labeled PG. The ability to induce lateral segregation of lipids was correlated to the translocation competency of the sequences in vivo [54]. The transmembrane orientation of the signal sequence in the membrane is similar to that of transmembrane segments of membrane proteins.…”