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Functional signal peptide reduces bilayer thickness of phosphatidylcholine liposomes
Y Tahara1, M Murata, S Ohnishi
1Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Abstract:
To investigate the interaction between a signal peptide and the lipid bilayer, two kinds of peptides, L8-M5 (L8 = MRL8PLAALG, M5 = KVFER) and L14-M5 (L14 = MRL14PLAALG), were examined in membranes composed of dioleoylphosphatidylcholine (DOPC). Peptides L8 and L14 are artificially designed signal sequences, and M5 is the N-terminal five residues of human lysozyme; L8 mediated effective secretion of human lysozyme in yeast, while L14 did not [Yamamoto, Y., et al. (1987) Biochem. Biophys. Res. Commun. 149, 431-436]. DOPC liposomes incorporating L8-M5 or L14-M5 were observed by electron cryomicroscopy as pairs of concentric circles, and the separation of the bilayer was measured along the membrane. Peptide L8-M5 was found to reduce the bilayer thickness, but L14-M5 did not. CD measurements revealed that L8-M5 adopted an alpha-helical conformation with random coil in the liposome membranes and that L14-M5 adopted a more helical and less random conformation than L8-M5. Fluorescence spectroscopy using both aqueous and membranous probes revealed that L8-M5 destabilized the lipid bilayer more strongly than L14-M5. These results suggest that functional L8-M5 reduces the bilayer thickness and destabilizes the lipid bilayer and that these activities are important for signal peptide function.
Insights
Functional signal peptides, like L8-M5, reduce lipid bilayer thickness and destabilize membranes. Non-functional L14-M5 did not exhibit these effects, suggesting their importance for signal peptide activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Biophysics
Background:
- Signal peptides mediate protein secretion across membranes.
- Artificial signal sequences L8 and L14 were designed, with L8 showing functional secretion in yeast.
- The interaction of these peptides with lipid bilayers is crucial for understanding their function.
Purpose of the Study:
- To investigate the interaction between two artificial signal peptides, L8-M5 and L14-M5, and dioleoylphosphatidylcholine (DOPC) lipid bilayers.
- To determine how these peptides affect membrane structure and stability.
- To correlate observed biophysical properties with signal peptide function.
Main Methods:
- Electron cryomicroscopy to visualize peptide-liposome complexes and measure bilayer separation.
- Circular dichroism (CD) spectroscopy to assess peptide secondary structure in membranes.
- Fluorescence spectroscopy using aqueous and membranous probes to evaluate lipid bilayer destabilization.
Main Results:
- L8-M5 significantly reduced the thickness of DOPC bilayers, while L14-M5 did not.
- L8-M5 adopted an alpha-helical conformation with some random coil in membranes, whereas L14-M5 was more helical.
- L8-M5 destabilized the lipid bilayer more effectively than L14-M5.
Conclusions:
- Functional signal peptide L8-M5 alters lipid bilayer thickness and enhances membrane destabilization.
- These biophysical changes induced by L8-M5 are likely essential for its role in signal peptide function.
- The study provides insights into the molecular mechanisms underlying signal peptide-membrane interactions.