Investigation of Transmembrane Helix Hetero-Dimerization by Combinatorial Peptide Libraries and High-Throughput Measurement of FÖRSTER Resonance Energy Transfer in Liposomes

Abstract

Receptor tyrosine kinases (RTKs) are membrane proteins containing an N-terminal extracellular ligand-binding domain, a single helical transmembrane (TM) domain, and an intracellular catalytic kinase domain. Lateral dimerization driven in part by the TM domain is proposed to be a crucial intermediate step in signal transduction across the plasma membrane. Defects in this process such as over-dimerization are closely linked to unregulated signaling and disease. Therefore, dimerization inhibitors developed using the chemical-physical basis of RTK TM domain dimerization could be promising therapeutic agents. Here we present a first step towards this goal. We designed a rational combinatorial peptide library based on the TM domain of Neu, an RTK from the Erb-B/HER epidermal growth factor receptor family (EGFR) with a pathogenic V to E mutation in the TM domain. Förster Resonance Energy Transfer (FRET) in lipid vesicles was used as a method to probe the dimerization between the library members and mutant Neu sequence. To enable high-throughput screening in liposomes, we developed a novel multi-well FRET assay. FRET donor-labeled library members and acceptor-labeled Neu peptides were incorporated into lipid vesicles in a rapid high-throughput 96-well plate format. We are using this high throughput screen to identify inhibitors of Neu dimerization. Our findings will be discussed in a context of throughput and sensitivity along with a statistical analysis that takes into account the FRET that arises from random proximity of donors and acceptors in the bilayer.