Membrane Protein Research Group, Department of Physiology and Department of Biochemistry, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada.
Biochemistry. 2010 Nov 2;49(43):9226-40. doi: 10.1021/bi101134h.
AE1, which exists in the erythrocyte plasma membrane as a noncovalent dimer, facilitates transmembrane Cl⁻/HCO₃⁻ exchange. Here a concatamer of AE1 (two AE1 monomers fused via a two-residue linker to form an intramolecular dimer) was designed to facilitate fluorescence resonance energy transfer (FRET) studies. The concatameric protein (AE1·AE1) was expressed at the plasma membrane at levels similar to that of wild-type AE1 and had Cl⁻/HCO₃⁻ exchange activity indistinguishable from that of wild-type AE1. Nondenaturing gel electrophoresis revealed that AE1·AE1 does not associate into higher-order oligomers when expressed in HEK293 cells and Xenopus laevis oocytes. The cysteine-less concatamer (AE1·AE1-C⁻) enabled introduction of unique cysteine residues into the whole intramolecular dimer. AE1(Q434C)·AE1(Q434C)-C⁻, with a single cysteine residue in each AE1 subunit, was labeled with the donor Alexa Fluor 488 C(5)-maleimide (AF) and the acceptor tetramethylrhodamine methanethiosulfonate (TMR-MTS). Energy transfer efficiency revealed that the distance between these residues in the AE1 dimer is 49 ± 5 Å. The 72% FRET efficiency observed between AE1(Q434C)·AE1-C⁻ labeled with AF and the lipid bilayer labeled with 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate indicates that Q434 is less than 33 Å from the lipid bilayer. We thus provide two distance constraints for the position of Q434, which is located in extracellular loop 1, connecting the first two transmembrane segments of AE1.
AE1 以非共价二聚体的形式存在于红细胞质膜中，促进跨膜 Cl⁻/HCO₃⁻交换。在这里，设计了一个 AE1 的串联体（通过两个残基的接头融合两个 AE1 单体形成分子内二聚体）以促进荧光共振能量转移（FRET）研究。串联蛋白（AE1·AE1）在质膜上的表达水平与野生型 AE1 相似，并且具有与野生型 AE1 相同的 Cl⁻/HCO₃⁻交换活性。非变性凝胶电泳显示，当在 HEK293 细胞和非洲爪蟾卵母细胞中表达时，AE1·AE1 不会形成更高阶的寡聚物。无半胱氨酸的串联体（AE1·AE1-C⁻）能够将独特的半胱氨酸残基引入整个分子内二聚体。AE1(Q434C)·AE1(Q434C)-C⁻，每个 AE1 亚基中都有一个半胱氨酸残基，用供体 Alexa Fluor 488 C(5)-马来酰亚胺（AF）和受体四甲基罗丹明甲硫磺酸（TMR-MTS）标记。能量转移效率表明，AE1 二聚体中这些残基之间的距离为 49 ± 5 Å。在用 AF 标记的 AE1(Q434C)·AE1-C⁻和用 1,1'-二癸基-3,3,3',3'-四甲基吲哚碳菁高氯酸盐标记的脂质双层之间观察到 72%的 FRET 效率表明，Q434 距离脂质双层小于 33 Å。因此，我们为 Q434 的位置提供了两个距离约束，Q434 位于连接 AE1 的第一和第二个跨膜片段的细胞外环 1 中。
