Analytical methods for the study of membranes and peptide-membrane interactions

Abstract

This thesis describes analytical work carried out to determine the stability and properties of lipid and peptide-lipid systems. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and tandem mass spectrometry (MSMS) analyses were carried out to establish a validated protocol for the complete identification of phospholipids, including the nature of the headgroup and acyl chains and the positions of the acyl chains on the glycerol backbone. Statistical differences were observed in the relative intensities of peaks corresponding to the neutral loss of the acyl chain from the sn-1 and sn-2 positions of POPE, POPC and OPPC, with a preferential cleavage of the chain from the sn-2 position of all three in the absence of added salt and a preferential cleavage at the sn-1 position in the presence of sodium or lithium ions. This knowledge was applied to the identification of unknown lipid mixtures both on the standard MALDI target plate and directly onto thin layer chromatography plates after separation. The above techniques, together with other analytical methods including thin layer chromatography and dynamic light scattering, were applied to the study and identification of lipids modified by actions such as hydrolysis and oxidation under conditions used for binding analyses of peptides and small molecules. Long-term analyses of samples containing synthetic melittin and liposomes showed that over time melittin both promotes the hydrolysis of liposomal lipids and is itself acylated. Analyses of the binding of a prototypical peptide (human neutrophil defensin HNP-2) to membranes, using methods that included dichroism and fluorescence spectroscopy, demonstrated that HNP-2 dimers act on lipid membranes via a carpet-type mechanism and allowed the rate of the formation of bound HNP-2 states to be determined. HNP-2 was modeled as a consecutive two-step process following pseudo-first order kinetics. The first step (membrane association) was rapid, with a half-life of around 0.5 minutes, while the second step (reorientation and partial insertion into the membrane) was slower by an order of magnitude