The effect of point mutations on the free energy of transmembrane α-helix dimerization¹

Abstract

Glycophorin A forms homodimers through interaction of the single, helical transmembrane domains of the monomers. The dimers are stable in sodium dodecylsulfate (SDS), permitting a number of studies that have identified a critical motif of residues that mediates dimer formation. We have used analytical ultracentrifugation to measure the energy of dimerization in a non-denaturing detergent solution and have observed the changes in energy arising from two of the mutants previously studied. Use of the detergent pentaoxyethylene octyl ether (C₈E₅) is a great advantage, since its micelles are neutrally buoyant and the detergent allows a reversible association to occur between monomer and dimer states of the glycophorin A transmembrane helices during the time-scale of sedimentation equilibrium. Use of this detergent in analytical ultracentrifugation may enable a wide range of studies of molecular association events in membrane proteins.

We find that the glycophorin A transmembrane helix dimerizes with a dissociation constant of 240(±50) nM, corresponding to a free energy of dissociation of 9.0(±0.1) kcal mol⁻¹. Point mutants that were found to be disruptive in SDS (L75A, I76A) reduced the dimer affinity in the C₈E₅ detergent environment (K_d = 1.7(±0.2) μM and 4.2(±0.9) μM, respectively). Thus, the earlier findings are placed on a quantitative, relative energy scale of association by our measurements. Molecular modeling and simulations suggest that the energy differences can be accounted for as changes in van der Waals interactions between helices.