Thus far, methods that give quantitative information about lateral interactions in membranes have been restricted peptides or simplified protein constructs studied in detergents, lipid vesicles or bacterial membranes. None of the available methods have been extended to complex or full length membrane proteins. Here we show how free energies of membrane protein dimerization can be measured in mammalian plasma membrane-derived vesicles. The measurements, performed in single vesicles, utilize the Quantitative Imaging FRET (QI-FRET) method. The experiments are described in a step-by-step protocol. The protein characterized is the transmembrane domain of Glycophorin A, the most extensively studied membrane protein, known to form homodimers in hydrophobic environments. The results suggest that molecular crowding in cellular membranes has a dramatic effect on the strength of membrane protein interactions.

Dysregulation of ligand-independent receptor tyrosine kinase (RTK) dimerization, which is the first step in RTK activation, leads to pathologies. A mechanistic understanding of the dimerization process is lacking, and this lack of basic knowledge is one bottleneck in developing effective RTK-targeted therapies. For instance, the roles and the relative contributions of the different RTK domains to RTK dimerization are unknown. Here we use quantitative imaging Förster resonance energy transfer (QI-FRET) to determine the contribution of the extracellular (EC) domain of fibroblast growth factor receptor 3 (FGFR3) to FGFR3 dimerization. We provide the first direct experimental evidence that the contribution of FGFR3 EC domain to dimerization is repulsive in the absence of ligand, and on the order of 1 kcal/mole. The magnitude of this repulsive contribution is similar to the dimer over-stabilization that can occur due to pathogenic single amino acid mutations, and therefore significant for biological function.

Intramembrane proteases cleave transmembrane substrates to liberate physiologically important molecules. The proteolytic activity of these enzymes can be significantly influenced by the composition of the lipid membrane. Here, we find that the composition of the lipid membrane has a dramatic effect on the motions of the GlpG rhomboid serine protease from Escherichia coli. In a 1-palmytoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer, conformational changes of two critical structural elements of the protease, the cap loop close to the active site and the regulatory loop L1, occur within 40ns of unconstrained molecular dynamics simulations. In contrast, in a 1-palmytoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) lipid bilayer, a conformational transition of the cap loop is observed only after~80ns,~20ns after that of loop L1. This sensitivity of the enzyme motions on the lipid membrane composition is explained by differences in how POPC and POPE lipid headgroups hydrogen bond among themselves, and with protein amino acids. Tight interactions between a lipid headgroup and the active site restrict the dynamics of the cap loop. An atomistic description of the structure and dynamics of the protease:substrate complex is a critical first step towards understanding how the protease works. Molecular dynamics simulations of GlpG together with the Spitz model substrate reveal that docking of the substrate to the enzyme involves a complex interplay of changes in the structure and dynamics of the substrate, the protease, and the surrounding lipid molecules.