Nanoscale spatial and temporal response of lipid membranes interacting with biomolecules and nanoparticles

Abstract

The cell membrane is the first point of interaction whenever any external biomolecule or nanoparticle encounters a cell. Thus the nature of interactions between the cell membrane and various proteins, nanoparticles, and drugs has been widely studied. But the cell membrane is very complex containing thousands of lipids and various types of membrane proteins. The simplification of the membrane to a model membrane is a necessity to understand the roles of the individual lipids in such interactions.

In the initial part of the thesis, the interactions between charged/uncharged NPs and multi-component model cell membranes have been discussed. Such interactions are governed by various factors like electrostatics, membrane stiffness, NP size, etc. The membranes segregate spatially into disordered and ordered micro-domains visible by microscopy but our X-ray study revealed the presence of nanodomains, which emulate real cell membrane heterogeneities. In our work, the stiffness of the membrane was varied by using two different types of zwitterionic lipids while the charge of the membrane was incorporated using a negatively charged lipid DPPG which forms the ordered phase of the membrane. The cationic NPs preferred to bind to the DPPG-rich nanodomains in the less stiff membranes and caused the nanodomains to coalesce. In the stiffer membranes, the cationic NP binding was more owing to the larger size of the DPPG-rich nanodomains with higher charge density. The dominance of electrostatics caused more binding of cationic NPs compared to the zwitterionic ones. Interestingly, increasing the NP concentration caused these to unbind from the membranes. The unbinding transition was dominated by electrostatics for the cationic NPs whereas it was mediated by crowding for the zwitterionic NPs

The next part of the thesis discusses the interaction between viral fusion peptides and model cell membranes. The human immunodeficiency virus (HIV) actively involves the glycoproteins containing the gp41 fusion peptide to fuse with the host membrane at the phase boundaries of the cholesterol-rich domains. Our study using fluorescence correlation spectroscopy (FCS) coupled with STED reveals the presence of hindered lipid diffusion at various nanoscale lengths in both the liquid-disordered (Ld) and the liquid-ordered (Lo) domains. The binding of the gp41 peptides altered the diffusion gradients across these length-scale dependent diffusion regimes in both domains, more pronounced in the Lo domains, also showing coalescence of nanodomains.

The last part of the thesis discusses the study of the interaction of a cyclic polypeptide named colistin, which is an antibiotic, with real E. coli bacterial membranes. The colistin is a membrane-targeting drug. Our STED-FCS analysis showed that the bacteria re-organized its membrane dynamics with increasing colistin doses and the final highly mutated E. coli presented a homogeneous type of diffusion at all length scales.

Overall, the thesis encompasses the role of cell membrane heterogeneities in playing a role in its interaction with various external entities and shows how the membrane structure, morphology, and dynamics are being altered at various length scales.