Membrane-Peptide Interactions Open Access
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The overarching goal of my research has been the investigation of peptide interactions with model membranes, how they affect membrane properties and vice versa. This dissertation focuses on two topics in this area: membrane interactions with the antimicrobial peptide, NA-CATH, and lipid diffusion in supported lipid bilayers. The main focus of my dissertation is characterizing antimicrobial peptides in the hopes of combating antibiotic resistance. This threat has prompted research into biological methods of combating bacterial infection. One such pervasive strategy employs cationic antimicrobial peptides, AMPs. These peptides use their structure to target and disrupt bacterial membranes. They specifically attach to PG (phosphatidylglycerol) and CL (cardiolipin) rich membranes, a common anionic lipid of the outer bacterial leaflet. Most have shown broad-spectrum activity, adequate potency, and minimal resistance. Considering these peptides have been active against pathogens for millions of years and have not developed any broad resistance, they are of particular interest to study. The focus of my membrane-antimicrobial peptide interactions studies is NA-CATH, which is found in the Naja atra snake. We have characterized the activity and behavior of this 34 amino acid AMP. Using one and two phase liposomes, we have found that lipid composition and the presence of phase separation affect the activity and behavior of NA-CATH. Fluorescence leakage and requenching assays indicate that the presence of phase separation increases leakage activity and the anionic lipid locale affects the leakage mechanism. The second focus of my dissertation is using fluorescence recovery after photobleaching, FRAP, to characterize lipid diffusion in supported lipid bilayers. Using this technique we have found that surface preparation affects the diffusion properties of the membrane. Additionally, we have explored the effects of cross-linking on lipid diffusion. Many cellular functions are initiated by the cross-linking and oligomerization of proteins on the membrane surface. For example, B cell receptors (BCRs) oligomerize upon antigen binding. The question remains of how these large clusters affect the surrounding lipids. In order to investigate this, our model consists of lipid bilayers with five mol % of the lipid headgroups biotinylated. Upon the addition of avidin, two biotinylated lipids are cross-linked together on the surface of the bilayer. We have found that avidin cross-linking does not influence lipid diffusion and mobility. However, it does cause anomalous subdiffusion and low mobility of the cross-linker. This is likely caused by the aggregation of avidin molecules on the surface of the bilayer.