Understanding the Organization and Dynamics of KRAS4b on a Complex 8-Lipid Reconstituted Model Membrane using Microscopy and Spectroscopy Methods

Abstract

The enrichment of negatively charged phospholipids, particular phosphatidylserine (PS) and phosphoinositides, in the inner leaflet of plasma membrane (PM) is one of the underlying principle behind recruitment of numerous intracellualr proteins to PM, thus regulating key cellular events. One such protein of interest is KRAS4b, a member of Ras superfamily of small GTPases, which is an oncoprotein implicated in 95% of pancreatic cancer, 45% of colorectal cancer, 35% of lung cancer and high extent in other forms of cancer. When Ras is active and bound to the membrane, it binds to its downstream effectors and triggers multiple signal cascading pathways that are essential for cell growth, proliferation and survival. KRAS4b localizes to PM predominantly by virtue of (i) the electrostatic interaction between six positively charged lysine residues located in the hypervariable region (C-terminal) of KRAS4b and negatively charged phospholipids in PM and (ii) the hydrophobicity of the farnesyl group, post-translational modification in C185 amino acid of KRAS4b, which anchors the protein into the disordered lipid domains. In this study, we aim to explore the molecular mechanism of KRAS4b interaction with the membrane using recombinant protein on an artificial supported lipid bilayer. The biomimetic surface is composed of complex 8-lipids composition that closely resemble the lipid composition of the inner leaflet of biological PM. We use a combination of fluorescence-based microscopy and single molecule spectroscopy techniques as well as high resolution scanning probe microscopy to study the lateral organization and dynamics of KRAS4b as well as the lipid bilayer. Using a very complex reconstituted model membrane to study KRAS4b, our research provides mechanistic insight into RAS biology and possibly a nobel platform for targeting RAS.