Quantifying the Dimerization Energy of a CLC Transporter in Lipid Bilayers

Abstract

Recently, a structurally stable and functional monomeric form of the normally homodimeric Cl−/H+ antiporter CLC-ec1 was designed by introducing two tryptophan mutations at the dimer interface, I201W and I422W. Several single tryptophan mutant constructs show intermediate stability between monomer and dimer state, as observed by size exclusion chromatography. In addition, the monomer and dimer populations can be shifted by adding lipids to the purified protein in detergent micelles, indicating that the system is undergoing reversible dimerization. We are now developing CLC-ec1 into a model for measuring dimerization energetics in a lipid bilayer. To measure the free energy, as well as enthalpic and entropic contributions, we must determine the fraction of monomer and dimer in the total protein population at different temperatures. To do this, we introduce cysteine residues into the extracellular loop adjacent to the dimerization interface, and fluorescently label the protein with tetramethylrhodamine- (TMR) or fluorescein-maleimide. The fluorescent protein is then reconstituted into liposomes at known concentration, and the fraction of dimer is measured in one of two ways. In the first method, protein is labeled with TMR at a position that allows the rhodamine molecules to undergo self-quenching by forming non-fluorescent pairs when in the dimer state. Addition of 0.5% SDS dissociates the dimer, as confirmed by glutaraldehyde cross-linking, leading to an increase in fluorescence at the rhodamine peak emission wavelength, and a measurement of the dimer population. In the second method, protein is co-labeled with fluorescein and TMR, and the Forster resonance energy transfer signal is measured from the dimer complexes. These studies introduce CLC-ec1 reversible dimerization as a simplified model for the thermodynamics of membrane protein folding and TM helix recognition in the membrane environment.