Description

Biological signaling is responsible for almost all cellular events that occur within an organism and its regulation is driven by signaling pathways. The MAPK/ERK signaling pathway has been extensively studied due to its medical relevance in cancer and its participation in crucial biological processes such as cell growth and apoptosis. A key regulatory step for this pathway is a cell membrane complex consisting of the proteins RAS GTPase and RAF kinase. RAS activates RAF by recruiting it onto the cellular membrane through specific binding events. Our lab seeks to explore the detailed molecular mechanism of RAF and the specific roles of regulatory domains during RAF activation using efficient fluorophores. Single-molecule experiments will allow us to determine different states during RAF recruitment. Visualization of proteins in fluorescent microscopy has been traditionally tagged with fluorescent proteins such as mNeonGreen, mNG. The use of fluorescent proteins as fluorophores can artificially interrupt the natural activity of proteins. For our fluorescent microscopic experiments, mNG’s large size may sterically hinder RAF and interrupt the binding to the membrane. The properties of mNG are not suitable to quantify the kinetic parameters of RAF due to its fast bleaching and size. In contrast to using mNG, we will use sortase labeling chemistry to introduce bright, organic dyes to a protein of interest. Sortase’s transpeptidase activity will be employed to covalently label RAF with a small organic dye, Alexa647. We will design, clone, and purify a RAF-dye protein construct and validate with single-molecule experiments in physiologically relevant conditions. Supported lipid bilayers, SLBs, are lipid bilayer platforms that mimic membrane environments that will be constructed to measure the binding of regulatory domains of RAF. There has been an emphasis on discovering molecular mechanisms of the MAPK/ERK pathway to pave the way for new pharmaceutical discoveries. The successful construction of RAF-Alexa647 will allow us to get one step closer to the discovery of possible therapeutics for MAPK/ERK-related diseases.