Investigating Membrane Dynamics And Oligomerization Of Pore-forming Toxins Using Single-molecule Fluorescence Techniques

Abstract

The biological membrane is a thin fluidic matrix composed of a lipid bilayer that forms the primary cellular barrier against the extracellular environment. The high-density of embedded proteins and glycosylated molecules confer further complexity with unique specificities for signalling and transport across the membrane. Many pathogenic bacteria have evolved dedicated proteins, pore-forming toxins (PFTs), to form nanoscale ring-like pores on cellular membranes that lead to cell lysis and death. However, it is challenging to study how PFTs function due to the considerable heterogeneity in their assembly intermediates and their complex interaction with lipid components. In this work, we have employed single-particle tracking and single-molecule photobleaching to investigate the assembly pathway of ClyA (a representative αPFT) on supported lipid bilayers (SLB). We show that cholesterol in the membrane greatly enhances the ClyA lytic activity by stabilizing the membrane inserted protomer intermediate and assisting in oligomerization by acting as a ’molecular glue’ between the protomer-protomer interfaces. We identify the role of different membrane-bound motifs of ClyA responsible for defining the initial membrane binding and the large conformational change required to form the pore. In the concluding part, we show how biomolecular assembly of PFTs can be enhanced in complex ways by crowded membrane surfaces using polyethylene glycol (PEG) grafted to lipids as crowders. As the PEG crowder transition from mushroom to an elongated polymer called brush regime, membrane-embedded molecules display correlated changes in their mobility and biomolecular assembly. Overall, this work elucidates how molecular and physical interactions modulate the biomolecular assembly of PFTs on lipid bilayer membranes.