The Significance of Surface Complementarity on the Free Energy of Membrane Protein Assembly in Membranes

Abstract

The dimerization interface of the 18-TM helix ClC-ec1 Cl-/H+ antiporter is formed by four alpha helices (H, I, P and Q) that are lined entirely by non-polar residues (leucines and isoleucines). The resultant surface fits well with its protein partner, presenting ClC-ec1 as an interesting model system to study the effect of shape complementarity on the stability of protein association. Recently, we established single-molecule fluorescence microscopy methods allowing for the measurement of the equilibrium dimerization reaction of ClC-ec1 in 2:1 POPE/POPG lipid bilayers. Here, we investigate the effect that cavities, engineered via subtractive substitutions to ALA, have on the free energy of dimerization in the lipid bilayer. We first engineered ClC constructs with large cavities by mutating entire helix surfaces to ALA, 4-5 residues at a time. Each of these constructs were stable and folded upon purification in detergent, and demonstrated comparable functional Cl- transport after reconstitution in membranes. However, while all-ALA helix I, P and Q are almost entirely dimeric as determined by size exclusion chromatography in detergent, all-ALA helix H shifts to a monomeric form. Further investigation of the residues on helix H indicates that L194A alone is responsible for the shift to the monomeric state. Cysteine accessibility and functional reconstitution experiments show that L194A is folded and functional in both monomeric and dimeric states. Measurement of the ΔΔG of dimerization in 2:1 POPE/POPG shows that L194A shifts stability by 2 kcal/mole, similar to the effect of a bulky I422W substitution at the interface. We conclude that L194 is an energetic hotspot on the dimerization interface, but the reason for this shift in stability remains unknown. Examination of the structure shows that L194 is positioned along the symmetry interface of the subunit fold suggesting that it may be involved in defining the shape of the dimerization interface, and thus mutation may alter shape complementarity by increasing dynamics of those helices.