The binding of melittin and the C-terminally truncated analogue of melittin (21Q) to a range of phospholipid bilayers was studied using surface plasmon resonance (SPR). The phospholipid model membranes included zwitterionic dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylethanolamine (DMPE), together with mixtures DMPC/dimyristylphosphatidylglycerol (DMPG), DMPC/DMPG/cholesterol and DMPE/DMPG. Melittin bound rapidly to all membrane mixtures, whereas 21Q, which has a reduced charge, bound much more slowly on the DMPC and DMPC/DMPG mixtures reflecting the role of the initial electrostatic interaction. The loss of the cationic residues also significantly decreased the binding of 21Q with DMPC/DMPG/Cholesterol, DMPE and DMPE/DMPG. The role of electrostatics was also highlighted with NaCl in the buffer, which affected the way melittin bound to the different membranes, causing a more uniform, concentration dependant increase in response. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high α-helicity was associated with high binding affinity. Overall, the results demonstrate that the positively charged residues at the C-terminus of melittin play an essential role in membrane binding, that modulation of peptide charge influences selectivity of binding to different phospholipids and that manipulation of the cationic regions of antimicrobial peptides can be used to modulate membrane selectivity.

Exosomes, a form of extracellular vesicle, are an important precursor in regenerative medicine. Microfluidic methods exist to capture these sub-micrometer sized objects from small quantities of sample, ideal for multiple...

The membrane interaction of the cyclotide kalata B1, an all-d-analogue and a single alanine substituted analogue (G6A), was studied by surface plasmon resonance (SPR) and atomic force microscopy (AFM). Kalata B1 showed a strong binding selectivity for dimyristoyl-phosphatidylethanolamine (DMPE) compared to dimyristoyl-phoshatidylcholine (DMPC)-containing lipids. However, when the interaction was visualized by AFM the peptide interacted with DMPC and DMPE in a similar manner. There was no apparent change in membrane morphology with either lipid, suggesting that kalata B1 does not act via a carpet-like disruption mechanism. The d-analogue showed similar binding by SPR and the same strong selectivity for DMPE, indicating that the membrane-interaction and lipid selectivity are not stereo-specific. SPR studies of the G6A analogue revealed that it interacted in a similar way to kalata B1 on the DMPC containing lipids, but showed no increased response on the DMPE containing lipids observed for kalata B1 and d-kalata B1. These results indicate that the Gly6 residue directly influences membrane binding as it is located near a putative membrane interacting hydrophobic patch. Overall, the data suggest that very small changes in amino acid composition (with no change in conformation) can influence specific self-association in combination with membrane binding and mediate the activity of kalata B1.

The chromatographic support containing monolayers of phospholipids offers novel modes in analyzing and separating proteins. The polar choline head groups on immobilized phosphatidylcholine were used for the affinity purification of phospholipase A (PLA). The purification process involves removing the contaminating proteins with detergent additives to the elution buffer such as short-chain alkylsulfonates. The lipid-bound PLA was eluted with acetonitrile or octyllysophosphatidylcholine. The purity of PLA was approximately 70% based on densitometric scans of gel electrophoresis. These results suggest that the lipid-immobilized chromatography may be applied to develop purification methods for PLA, enzymes, and membrane proteins obtained from diverse cells.