title: The effect of point mutations on the free energy of transmembrane alpha-helix dimerization.
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 (Kd=1.7(+/-0.2) microM and 4.2(+/-0.9) microM, 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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114, USA.
authors.forename: K G
authors.initials: KG
authors.lastname: Fleming
authors.forename: A L
authors.initials: AL
authors.lastname: Ackerman
authors.forename: D M
authors.initials: DM
authors.lastname: Engelman
mesh_terms: D002462:Cell Membrane / Q000737:chemistry; D003198:Computer Simulation; D003902:Detergents; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D006021:Glycophorins / Q000737:chemistry* / Q000235:genetics; D008823:Micelles; D017354:Point Mutation / Q000502:physiology*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins; D013816:Thermodynamics; D014461:Ultracentrifugation / Q000379:methods*
chemical_list: D003902:Detergents; D006021:Glycophorins; D008823:Micelles; D011993:Recombinant Fusion Proteins