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首页> 外文期刊>Journal of Molecular Biology >THE EFFECT OF POINT MUTATIONS ON THE FREE ENERGY OF TRANSMEMBRANE ALPHA-HELIX DIMERIZATION
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THE EFFECT OF POINT MUTATIONS ON THE FREE ENERGY OF TRANSMEMBRANE ALPHA-HELIX DIMERIZATION

机译：点突变对跨膜α-螺旋二聚自由能的影响

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摘要

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)mu M and 4.2(+/-0.9)mu M, 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. (C) 1997 Academic Press Limited. [References: 33]
机译：糖皮质激素A通过单体的单个螺旋跨膜结构域的相互作用形成同型二聚体。二聚体在十二烷基硫酸钠（SDS）中稳定，因此允许进行许多研究，这些研究确定了介导二聚体形成的残基的关键基序。我们已使用分析超速离心法测量了非变性洗涤剂溶液中的二聚化能量，并观察到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）具有很大的优势，因为其胶束呈中性浮力，并且去污剂允许在沉淀平衡的时间范围内，糖蛋白A跨膜螺旋的单体和二聚体状态之间发生可逆结合。在分析超速离心中使用这种去污剂可以对膜蛋白中的分子缔合事件进行广泛的研究。我们发现，糖蛋白A跨膜螺旋二聚体具有240（+/- 50）nM的解离常数，对应于9.0（+/- 0.1）kcal mol（-1）的解离自由能。发现在SDS中具有破坏性的点突变体（L75A，I76A）在C8E5去污剂环境中降低了二聚体亲和力（分别为K-d = 1.7（+/- 0.2）μM和4.2（+/- 0.9）μM）。因此，通过我们的测量，较早的发现被置于定量的相对能量关联度上。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。 （C）1997 Academic Press Limited。 [参考：33]

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