Effect of LS3 peptide on the structure of Dioleoylphosphatidylethanolamine lipid membrane formations

In an attempt to understand the anti-bacterial action of LS3, a synthetic helical peptide (LS3), the interaction of the peptide with a non-lamellar forming lipid dioleoylphosphatidylethanolamine (DOPE) was studied. Using Differential Scanning Calorimetry (DSC) it was found that the introduction of LS3 at increasing concentrations decreased the phase transition temperature of the DOPE lipid from the lamellar to inverse hexagonal phase by 3°C (Sa’adedin, Bradshaw., 2010). Further studies on the effect that LS3 concentration had on the DOPE lipid system were conducted using Small Angle X-ray diffraction at the ELETTRA Synchrotron in Trieste, Italy. The introduction of LS3 on the DOPE membrane showed that the peptide actively induced or stabilised a number of non-biological lipid structures. In particular, it was clearly shown that two different structures were formed and stabilised in the presence of the peptide over a wide range of temperatures. The level of destabilisation of the biologically stable phase was found to be substantial, with increasing concentration of peptide in the membrane almost eliminating the lamellar phase entirely in the sample. The two different structures that were formed as a result of the introduction of the LS3 peptide varied in size depending on the concentration of LS3 peptide introduced. LS3 stabilises the inverse hexagonal phase and cubic phases, by reducing the negative curvature stress of these structures, and at high concentrations of LS3 the size of cubic and inverse hexagonal structure is reduced. This can be explained by: the water being removed from the surrounding lipids, resulting in a more 3 tightly packed inverse hexagonal structure. This is an unexpected result, as LS3 is a peptide that has associated water with it due to the serine amino acids present. Lastly, I studied the positioning of the LS3 peptide on DOPE bilayers at a molecular level. In order to do this, I used the Membrane Diffractometer (D16) at the Institut Laue-Langevin (ILL) in Grenoble. Testing at two separate LS3 concentrations, I used deuterated labelled LS3 peptide (a heavy Hydrogen) in order to identify the positioning and orientation of the peptide on the DOPE bilayer system. It was found that, at both concentrations, the peptide resided at the lipid/water interface of the membrane at an angle between 4°-6°. The insertion of the peptide into the lipid, disrupts bilayer stability. The current model in peptide/membrane destabilisation, states that membrane destabilisation correlates to the depth of insertion of a peptide in a bilayer. My results show that the depth of a peptide in the membrane is not required for increased membrane curvature/membrane destabilisation at the lamellar phase as LS3 was located at the lipid/water interface.