Nanolipoproteins (NLP) systems allow transmembrane proteins and receptors to be studied in their near-native environment (TRD1.3). To access the function of receptor proteins, we are constructing in vitro model membranes with embedded receptor proteins. We also develop new approaches to tune the size of these membranes, and integrate fluorescent reporters into the model membrane systems. The fluorescent reporters are integrated via click chemistry to enable the monitoring of signaling dynamics with high spatial and temporal resolution. Our aim is to establish versatile model membrane systems that are larger and easier to produce than previous systems to enable in vitro studies of the multi-protein structures required for signaling.
Expression of epidermal growth factor receptors in membrane nanodiscs
Membrane proteins are 60% of drug targets, yet mechanistic studies of membrane proteins have been particularly challenging due to the difficulty of maintaining a physiological environment during experiments. In recent years, the use of model membrane technologies, in particular nanolipoproteins (NLPs), has exploded because they provide near-physiological environments for membrane proteins while allowing access to both the intra- and extracellular domains. Because of these advantages, NLPs enable a range of structural and functional assays. We improve the size tunability of the membrane discs and develop strategies to integrate fluorescent reporters. Through this combination, we create a platform that enables single-molecule Förster Resonance Energy Transfer (smFRET) measurements to push our mechanistic understanding of membrane proteins.
Of particular interest is the epidermal growth factor receptor (EGFR). EGFR, a canonical example of receptor tyrosine kinases, plays a significant role in signaling leading to cell survival, growth and differentiation [1]. Overexpression of EGFR or mutations in EGFR cause several diseases including cancer [2]. EGFR binds to seven different ligands in its extracellular domain and transmits this information to the intracellular domain [3]. To study signal transduction of EGFR across the membrane, we express full-length EGFR in membrane nanodiscs using cell-free expression. The intracellular domain of EGFR is fluorescently labeled with snap surface 594 (ss594) [4].
To characterize the changes in conformation of the intracellular domain upon ligand binding to the extracellular domain, we introduce a small percentage of cy5 labeled PE lipids in the nanodiscs as illustrated in Fig. 1. ss594 dyes act as donors and cy5 dyes in the membrane behave as acceptors for sm-FRET measurements. Using the technique of nanolipoproteins and sm-FRET, we aim to map out the spatiotemporal readouts of the receptor in response to ligand binding and signaling.