Measuring the Free Energy of ClC-ec1 Dimerization in Membranes using Single Molecule Photobleaching Analysis

Abstract

Why do greasy membrane proteins interact with their greasy protein partners instead of the similarly greasy lipid bilayer? This is a question at the root of membrane protein oligomerization and folding in membranes. Recently, we turned to the homodimeric ClC-ec1 Cl-/H+ antiporter, in order to develop a new model system that can be used to investigate this question. The ClC-ec1 dimerization interface is large (1200 A2) and lined by ∼20 non-polar residues, forming an interaction surface exhibiting high shape complementarity. Previous studies showed that ClC-ec1 is folded and functional in both monomeric and dimeric states. Using a single-molecule microscopy approach, we measured the photobleaching distributions across a wide range of subunit/lipid dilutions, with the lowest representing a sub-biological condition of 1 subunit per 40 E. coli membranes. The data shows that wild-type ClC-ec1 follows a monomer to dimer reaction that is reversible and fits to an equilibrium isotherm, allowing for determination of the dimerization free energy in 2:1 POPE/POPG lipid bilayers (Chadda et al., eLife 2016). Next, two approaches were used to investigate how residues at the interaction interface influence the free energy of dimerization. First, bulky TRP residues were introduced resulting in a destabilization of the dimer by 2 kcal/mole per TRP. Second, subtractive substitutions to ALA were constructed introducing cavities at the interface. While most of the ALA substitutions appear to have no effect, L194A destabilizes the dimer by 2 kcal/mole in lipid bilayers while maintaining both fold and function. These studies present ClC-ec1 as a robust model system for probing the physical forces driving protein association in membranes.