Dimerization of rhomboid protease RHBDL2 in lipid membranes addressed by FRET with MC simulations

Many membrane proteins are thought to function as oligomers, but measuring membrane protein dimerization in native lipid membranes is particularly challenging. Forster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are non-invasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. The effects inherent to such two-dimensional systems, such as excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and FCS we introduce methods to measure surface protein density and to estimate kappa squared, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined 2D environments. We then use FRET and FCS to analyze the dimerization of human rhomboid protease RHBDL2 in its native lipid membranes. While previous reports have proposed that rhomboid proteases dimerize and this allosterically activates them, we find no evidence for stable oligomers of RHBDL2 in lipid membranes of human cells. This indicates that the rhomboid transmembrane core may be intrinsically monomeric. Finally, our findings will find use in the application of FRET and FCS for the analysis of oligomerization of transmembrane proteins in lipid membranes.