Investigation of cell membrane dynamics A potential marker for lipid response towards membrane active proteins and peptides

Abstract

The cell membrane is made up of lipids and proteins held together with intermolecular hydrophobic/hydrophilic interactions. These physical non-covalent interactions help in maintaining the integrity of the cell membrane, yet allowing them to have a flexible and fluidic nature, exploited by the cell to cr ntro1 the transport of nutrients and cell signaling events. The interaction between a membrane targeting molecule and lipid bilayer is a crucial step when a pathogen tries to infect a host cell. Likewise, the cell envelope of most virulent pathogens is the primary target for common drug molecules and antibiotics. Understanding the underlying physical interactions between membrane targeting molecules of both prokaryotic and eukaryotic cell membranes is an important aspect of developing strategies to mitigate virulent diseases. In this thesis, I will emphasize the importance of understanding the dynamics of membrane lipids, which provides a unique marker to identify and characterize protein/peptide-lipid interaction. In the first part, I have described our results on the interaction of membrane targeting pore-forming toxin, listeriolysin O (LLO) used by the bacteria Listeria monocytogenes to attack and disrupt the host cellular machinery. LLO oligomerizes upon binding to the lipid bilayer forming either complete or incomplete rings that are observed to exist in an inserted transmembrane pore state or an un-inserted pre-pore state. The significant outcome from this part of the study is our ability to identify the signatures of different structural states of LLO, which have a distinct effect on lipid bilayer diffusivity. Using vesicle leakage as an indicator of pore function in combination with insights regarding membrane-bound structure from Förster resonance energy transfer (FRET) and lipid dynamics from fluorescence correlation spectroscopy (FCS) measurements on single giant vesicles exposed to LLO, we found that the un-inserted state of LLO decreases the lipid mobility whereas the inserted state enhances the sam