Development of a Novel FRET Assay to Characterize the Oligomerization State of Self-Associating Transmembrane Helices

Abstract

Using genetic reporter assays, significant self-interaction has been discovered in over 50 transmembrane (TM) helices from single-pass membrane proteins. To understand the biological functions of these TM interactions, which include signal transfer across the membrane in cancer-related pathways, it is necessary to construct accurate structural models of the TM oligomer. However in most cases this is not possible because the oligomerization state (stoichiometry) is unknown. It is often assumed that the helices form a dimer, though biologically relevant TM oligomers include dimers, trimers, and pentamers, as exemplified by GpA, HLA II Ii, and phospholamban, respectively.To characterize the stoichiometry of interacting single-pass membrane proteins, we are establishing a novel method based on Forster resonance energy transfer between identical fluorophores (homo-FRET). In order to allow the rapid bacterial expression and the purification of a wide range of fluorophore-coupled TM helices, we have developed TM fusion proteins with green fluorescent protein (GFP) and affinity tags. The measured homoFRET (via steady-state anisotropy) is proportional to both the affinity of the helices and the oligomerization state. Using only a fluorescence microplate reader, we demonstrate significant homo-FRET-coupling for GFP fusion proteins. Theoretical studies have shown that gradual photobleaching should yield a pattern of polarization that depends on the oligomerization state. High-profile experimental studies have confirmed this in-vivo using advanced microscopy. We show for the first time that the “fingerprint”-like curves after laser photobleaching can be achieved in-vitro with relatively simple equipment, allowing us to differentiate between oligomerization states. This demonstrates the feasibility of a rapid homo-FRET assay to determine the stoichiometry of transmembrane proteins in liposomes.