Perturbations of Cellular Model Membranes Induced by Membrane Interacting Model Peptide, Protein and Proteoliposome

Abstract

The cell membrane interface constantly encounters a highly crowded environment of membrane interacting biomolecular entities present, extracellularly and intracellularly, that both influence as well as exploit the physicochemical properties of membrane to execute key cellular functions. This thesis is majorly focused on the perturbation of the cellular membranes induced by three physiologically important membrane interacting model biomolecular entities, namely - Nisin (an antimicrobial peptide), a Hepatitis E virus-like particle (a globular viral coat protein) and bacterial membrane vesicle (secreted by E. coli).
Nisin is a 34-amino acid residue long peptide known to inhibit Gram positive bacteria viability by interacting with lipid II lead to pore formation and restriction of cell wall synthesis. Our findings suggest that nisin can inhibit Gram positive B. subtilis and also Gram negative E. coli bacteria (lipid II deprived) in high concentration regime. Using experimental and molecular dynamics simulations, we shown that high concentration regime of nisin can non-specifically interact with phospholipids and deform membrane in a lipid-II independent manner mechanism that depends on surface density and degree of peptide oligomerization. Furthermore, we dissect the attenuating role of nisin on neuroblastoma cell growth as observed by MTT assay. The underlying mechanism of attenuation of cancer cell growth was due to fluidizing effect of nisin on cancer cell membrane verified by decrease in anisotropy and in-plane elasticity of cell membrane.
The second model biomolecule, a globular viral coat protein Hepatitis E virus like particle (HEV-LP) was chosen which infects liver cells and we focussed on the entry mechanism through hepatic cell membrane. The binding and passive entry of HEV-LPs in hepatic cell model membrane was confirmed by the fluorescence microscopy and observed significant change in dipole potential, membrane fluidity and non-ideal mixing with hepatic cell model membrane. Specifically, lipids containing anionic lipid headgroups i.e. DOPS, DOPG and Liver PI and DOPE critical for the binding and membrane internalization of HEV along with low cholesterol content in membrane. Together, our findings suggest that the changes in the host cell membrane mechanical properties induced by HEV-LP crowding might facilitate virus penetration through host cell membranes.
The third biomolecular model system was proteoliposome - bacterial membrane vesicles (MVs) secreted by E. coli which facilitate long-distance delivery of bacterial virulence factors crucial for pathogenicity. Whether MVs modulate the physicochemical properties of xv
the host lipid membrane remains unknown. We quantitatively show that MV interaction increases the fluidity, dipole potential and elasticity of a biologically relevant multi-component host model membrane. Such modulation is facilitated by the presence of lipids containing head-groups such as phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol as well as a moderate acyl chain length of C16. While significant binding of MVs to the raft-like lipid membranes with phase separated regions of the membrane was observed, however, the elevated levels of cholesterol tend to hinder the interaction of MVs.
Together, this thesis attempts to understand a broader picture of how cellular membranes respond to perturbation induced by biomolecular entities of diverse shapes and biochemical nature that are often encountered by the biological membrane interface. We find that phase boundary conditions, line tension, negatively charged lipids are some of the common parameters that come into play during membrane interaction of structurally different biomolecules, further, degree of membrane perturbance may be dependent on the magnitude of molecular dipoles and curvature of the interacting biomolecules.