THE EFFECT OF POINT MUTATIONS ON THE FREE-ENERGY OF TRANSMEMBRANE ALPHA-HELIX DIMERIZATION

Glycophorin A forms homodimers through interaction of the single, heli cal transmembrane domains of the monomers. The dimers are stable in so dium dodecylsulfate (SDS), permitting a number of studies that have id entified a critical motif of residues that mediates dimer formation. W e have used analytical ultracentrifugation to measure the energy of di merization in a non-denaturing detergent solution and have observed th e 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 sta tes of the glycophorin A transmembrane helices during the time-scale o f sedimentation equilibrium. Use of this detergent in analytical ultra centrifugation may enable a wide range of studies of molecular associa tion events in membrane proteins. We find that the glycophorin A trans membrane helix dimerizes with a dissociation constant of 240(+/-50) nM , corresponding to a free energy of dissociation of 9.0(+/-0.1) kcal m ol(-1). Point mutants that were found to be disruptive in SDS (L75A, I 76A) reduced the dimer affinity in the C8E5 detergent environment (K-d =1.7(+/-0.2)mu M and 4.2(+/-0.9)mu M, respectively). Thus, the earlier findings are placed on a quantitative, relative energy scale of assoc iation by our measurements. Molecular modeling and simulations suggest that the energy differences can be accounted for as changes in van de r Waals interactions between helices. (C) 1997 Academic Press Limited.