‘Forbidden’ States Boost Fluorescence Resonance Energy Transfer (FRET) Studies of Membrane Receptors in Live Cells

Abstract

For probing biomolecular interactions or conformational changes in a live-cell setting the distance depending Fluorescence resonance energy transfer (FRET) is often the method of choice. The G-protein-coupled receptor (GPCR) superfamily mediates cellular responses and communication across cellular membranes, and is the largest known class of molecular targets with proven therapeutic value. On the one hand, real-time monitoring of membrane receptors and their interactions is vital to understand how they act in concert. On the other hand, the design of GPCR FRET probes is crucial to ensure unhampered functionality and binding kinetics of the molecular complex. Thus, such FRET probes usually require labeling compromises concerning donor-acceptor positions, distances and orientations, which limit the detectability of emitter signals in general, and the dynamic range of the FRET changes in particular. Here, we present an approach to optimize the energy transfer without changing the design of the FRET probe. We show that gold coated glass cover slips allow reinforcing the otherwise forbidden donor-acceptor energy transfer by virtual optimization of the dipole orientation. First, we confirm the resulting enhanced FRET on our nano-coatings for the inactive M1 muscarinic acetylcholine receptors (mAChRs) labeled with a CFP-FlAsH pair in classical bleaching experiments. Second, we demonstrate the advantage of this enhanced FRET technique for ligand binding studies in live cells by the increased dynamic FRET response between the inactive and active state of the M1 mAChR receptor. We believe that our techniques has particular potential for pharmaceutical drug screening to boost non-ideal FRET probes, and thus amplify signal responses without interfering with the well-characterized molecular assay.