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 wild-type and revertant signal peptides predominantly formed alpha-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 alpha-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.
Highlights
Targeting of the newly synthesized proteins in the cytoplasm of Escherichia coli is determined by signal peptides attached to the N-terminal end of the mature proteins [1]
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
This suggests that the functional signal peptide promotes the phospholipid clustering in lipid bilayers, thereby inducing the formation of domains enriched with PG
Summary
Materials—All phospholipids and NBD-PG were purchased from Avanti Polar Lipids (Birmingham, AL) and used without further purification. Each concentration of peptide and phospholipid was measured to determine the peptide/lipid (P/L) molar ratio. Except for measuring the reconstituted amount of signal peptides into liposomes, all experiments were undertaken using samples without the salt extraction step. To measure fluorescence anisotropy (A), 0.5 mol% DPH was included in liposomes. The concentrations of fluorescent probes were determined spectrophotometrically at 342 nm using 38,000 cmϪ1 for pyrene-PG, at 350 nm using 88,000 cmϪ1 for DPH, at 465 nm using 22,000 cmϪ1 for NBD-PG (and NBD-PE), and at 500 nm using 80,000 cmϪ1 for BODIPY-PG as the molar extinction coefficients. When the excimer and monomer fluorescence of pyrene-PG were measured the excitation wavelength was 342 nm, and the emission was in the range of 360 –500 nm. Iʈ and IЌ are the intensities measured with polarizers parallel and perpendicular to the vertically polarized exciting beam, respectively
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