Fleming K G, Ackerman A L, Engelman D M
Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114, USA.
J Mol Biol. 1997 Sep 19;272(2):266-75. doi: 10.1006/jmbi.1997.1236.
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.
血型糖蛋白A通过单体的单个螺旋跨膜结构域之间的相互作用形成同型二聚体。这些二聚体在十二烷基硫酸钠（SDS）中是稳定的，这使得许多研究能够确定介导二聚体形成的关键残基基序。我们使用分析超速离心法来测量在非变性去污剂溶液中二聚化的能量，并观察了先前研究的两个突变体引起的能量变化。使用去污剂五氧乙烯辛基醚（C8E5）具有很大优势，因为其胶束具有中性浮力，并且在沉降平衡的时间尺度内，该去污剂能使血型糖蛋白A跨膜螺旋的单体和二聚体状态之间发生可逆缔合。在分析超速离心中使用这种去污剂可能有助于对膜蛋白中的分子缔合事件进行广泛研究。我们发现血型糖蛋白A跨膜螺旋以240（±50）nM的解离常数二聚化，对应于9.0（±0.1）kcal mol-1的解离自由能。在SDS中被发现具有破坏作用的点突变体（L75A、I76A）在C8E5去污剂环境中降低了二聚体亲和力（Kd分别为1.7（±0.2）μM和4.2（±0.9）μM）。因此，我们的测量将早期的发现置于定量的、相对能量的缔合尺度上。分子建模和模拟表明，能量差异可以解释为螺旋之间范德华相互作用的变化。
