Unraveling the Mystery of Three-State Diffusion Model of KRAS4b on Plasma Membrane

Biological membranes are heterogeneous, complex and dynamic structures that regulate the conformation, function and dynamics of membrane proteins involved in cell signaling, survival and proliferation. KRAS4b, a small GTPase, is one such membrane protein. KRAS4b, when bound to the membrane and in an “active” GTP-loaded state, binds to several downstream effectors and triggers several signaling pathways. Importantly, membrane tethering of KRAS4b is an absolute requirement for its activity. Several oncogenic mutations in KRAS4b lock the protein in the active state leading to uncontrolled cell proliferation and eventually cancer growth. Therefore, understanding the complex biophysical nature of KRAS4b on the membrane may reveal a new frontier in finding therapeutic targets. Recent studies show that KRAS4b dynamics can be explained by an isoform specific 3-state diffusion model with a unique inter-state transition path in the inner leaflet of the plasma membrane and is highly regulated by the local lipid composition proximal to the protein. However, since the cellular plasma membrane is complex and experiments are limited by the detection limit of imaging technology, it is a challenge to understand the driving biophysical mechanisms behind each diffusion state. Using single particle tracking studies combined with statistical analysis methods, we attempt to unravel the underlying reasons for the three-state diffusion behavior of KRAS4b on the plasma membrane. We measured the diffusion of fluorescently labeled full length and fully processed KRAS4b (farnesylated and methylated) on reconstituted supported lipid bilayer of various compositions ranging from simple 2-lipids up to more complex 8-lipid systems. We show that the three- state diffusion model of KRAS4b is primarily driven by membrane composition and the mobility rates are highly regulated by membrane electrostatics and complexity as well as protein clustering on the membrane.