Measuring Large Membrane Protein Dimerization in Lipid Bilayers by Forster Resonance Energy Transfer

Abstract

Our understanding of the thermodynamics forces in membrane protein assembly in a lipid bilayer is limited, because there are not many equilibrium models for studying protein association in lipid bilayers. We have developed a new model system based on the homodimeric CLC-ec1 Cl−/H+ antiporter (WT) that offers an opportunity to investigate this. A single tryptophan substitution (I422W) in WT yields a monomer/dimer mixture in detergent. We quantitatively labeled CLC-ec1 with Cy3 donor and/or Cy5 acceptor to measure Forster resonance energy transfer (FRET) changes upon dimerization. By single-molecule TIRF microscopy we measured the molecular FRET efficiency of as 0.7 ± 0.1 (n=3). We then measured the population FRET efficiency of I422W in large membranes while varying the protein/lipid fraction (χ) to determine a Kd of dimerization. Emission spectra obtained from the fluorometer is fit using an in-house software Fytt to decompose contributions from the sensitized emission of donor, acceptor, FRET and background and to quantify FRET efficiency (E). We determined E for the positive dimer control by co-labelling with Cy3/Cy5 across a range of χ. Measuring I422W-Cy3 + I422W-Cy5 in membranes shows low E at low χ that increases and saturates to all-dimer E at high χ, indicating a dimerization reaction in membranes. Furthermore, we observe reversibility of the reaction when going from a high χ to low χ by diluting all-dimer I422W membranes with empty lipid bilayers, and the appearance of FRET upon fusion of separately labelled I422W-Cy3 with I422W-Cy5 membranes, indicating subunit exchange. We compared 2:1 POPE/POPG vs. POPC lipid compositions, and while both show a similar reaction, the POPC data fits well to a dimerization isotherm with Kd(I422W) = 4 x 10−6 protein/lipid, suggesting that we are measuring equilibrium dimerization reaction in membranes.