Adair B D, Engelman D M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520.
Biochemistry. 1994 May 10;33(18):5539-44. doi: 10.1021/bi00184a024.
Glycophorin A and its isolated transmembrane region (GpATM) are each known to form sequence-specific dimers in SDS micelles. Whether this behavior accurately reflects behavior in red cell membranes or lipid bilayers, however, has remained unclear. Resonance energy transfer between labeled GpATM peptides has been used to observe dimerization of GpATM in bilayers. Separate populations of GpATM peptides were labeled with 2,6-dansyl chloride as the donor chromophore and dabsyl chloride as the acceptor. Quenching of the 2,6-dansyl chloride by the dabsyl group demonstrated an association of the labeled peptides. The quenching was not affected by increases in the amount of lipid present or by unlabeled heterologous peptides but was decreased by the addition of unlabeled GpATM. GpATM was determined to form dimers by fitting the observed energy transfer for a number of donor to acceptor ratios and fitting to the expected number of donor labeled peptides in an oligomer with an acceptor as a function of oligomer number. The finding that the GpATM peptide forms helical dimers in lipid bilayers supports the idea that GpA is a dimer in the erythrocyte membrane. The resonance energy transfer approach may extend to the study of other oligomeric complexes.
已知血型糖蛋白A及其分离的跨膜区域（GpATM）在十二烷基硫酸钠（SDS）胶束中均能形成序列特异性二聚体。然而，这种行为是否准确反映了其在红细胞膜或脂质双层中的行为，仍不明确。标记的GpATM肽之间的共振能量转移已被用于观察GpATM在双层中的二聚化。分别用2,6-二甲基氨基萘磺酰氯作为供体发色团和二甲基氨基苯磺酰氯作为受体对不同群体的GpATM肽进行标记。二甲基氨基苯磺酰基团对2,6-二甲基氨基萘磺酰氯的淬灭表明了标记肽之间的缔合。淬灭不受脂质含量增加或未标记的异源肽的影响，但添加未标记的GpATM会使其降低。通过拟合多个供体与受体比例下观察到的能量转移，并根据寡聚体数量拟合寡聚体中预期的供体标记肽数量，确定GpATM形成了二聚体。GpATM肽在脂质双层中形成螺旋二聚体这一发现支持了血型糖蛋白A在红细胞膜中是二聚体的观点。共振能量转移方法可能会扩展到对其他寡聚复合物的研究。
