Pore Formation and Dynamics of Cytolysin-A on Supported Lipid Bilayer Membranes

Abstract

Ayush Agrawal1, Rajat Desikan1, Pradeep S.3, K. G. Ayappa1, J. K. Basu2, Sandhya Visweswariah41 Department of Chemical Engineering, Indian Institute of Science2 Centre for Condensed Matter Theory, Department of Physics, Indian Institute of Science3 Bioengineering program, Indian Institute of Science4 Department of Molecular Reproduction, Development and Genetics, Indian Institute of ScienceObserving the structure and dynamics of large protein assemblies on membranes has implications on understanding the general principles behind protein aggregation and engineering drug therapies. Dynamic and structural data on mesoscopic length and time scales are scarce for these systems. Cytolysin-A (ClyA) is a pore forming toxin that is known to cause unregulated pores on various cell membranes. In order to study pore formation of ClyA on model membranes, supported lipid bilayers (SLBs) of DMPC were incubated with varying concentrations of ClyA. Pore densities were quantified using atomic force microscopy (AFM) and led to the observation that a threshold toxin concentration is necessary for pore formation. The influence of the bilayer preparation protocol was observed by contrasting the pore densities on SLBs prepared using Langmuir-Blodgett (LB) deposition as well as vesicle deposition. Pore formation at varying deposition pressures on different substrates (mica, silicon) were investigated using LB prepared bilayers. Interestingly, at a high toxin concentration of 8.82 nM (large pore density regime), membrane undulations were prominently observed in the AFM images. Understanding the lipid dynamics around protein assemblies has implications on protein signalling, stability and function. Fluorescence imaging using FRAP and FCS on SLBs incubated with ClyA, and complementary molecular dynamics simulations were carried out to investigate the inherent heterogeneity in lipid dynamics in the vicinity of the ClyA pore.