Observing transmembrane conformational signaling with smFRET.

Abstract

Transmembrane receptors transmit information about the local chemical environment across the membrane to regulate cellular signaling. The conformational changes responsible for signal transduction have been inaccessible due to the challenges associated with isolating and interrogating full-length receptors. As a result, studies have been limited to individual domains, where the transmembrane conformational dynamics are inherently missing. We overcame these limitations by embedding full-length receptors within membrane nanodiscs, a lipid bilayer encircled by a solubilizing belting protein, and using single-molecule Förster resonance energy transfer (FRET) to follow the transmembrane conformational changes through the receptor. First, in the aspartate chemoreceptor from E. coli we find a change in the cytoplasmic helical packing and dynamics upon ligand occupancy, which may be a general mechanism in coiled-coil signaling proteins. Second, in the mammalian epidermal growth factor receptor (EGFR), we identify a ligand- and lipid-dependent conformational change of the intracellular domain upon extracellular ligand binding, revealing a previously unknown transmembrane conformational coupling that, remarkably, is mediated by a single transmembrane alpha-helix.