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首页> 外文期刊>Biochemistry >Conformational changes in the cytoplasmic domain of human anion exchanger 1 revealed by luminescence resonance energy transfer.
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Conformational changes in the cytoplasmic domain of human anion exchanger 1 revealed by luminescence resonance energy transfer.

机译：通过发光共振能量转移揭示人阴离子交换剂1的胞质结构域的构象变化。

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摘要

The cytoplasmic domain of the human erythrocyte anion exchanger 1 (cdAE1) serves as a center of organization for the red blood cell cytoskeleton as well as several metabolic enzymes and hemoglobin. The protein is known to undergo a reversible pH-dependent conformational change characterized by a 2-fold change in the intrinsic fluorescence and an 11 A change in the Stokes radius. While the exact changes in the molecular structure are unknown, on the basis of the crystal structure of the protein at pH 4.8 and site-directed mutagenesis studies, Zhou and Low (19) have proposed that the peripheral protein binding (PPB) domain of cdAE1 moves away from the dimerization domain in response to increasing alkalinity. To test this hypothesis, we have applied luminescence resonance energy transfer (LRET) to measure the intermonomer distance between donor and acceptor probes at the Cys(201) site (located in the PPB domain) within the cdAE1 dimer. This distance was found to increase as the pH is increased from 5 to 10, in recombinant forms of both the wild type and a mutant (C317S) of cdAE1. Furthermore, LRET measurements in red blood cell inside-out vesicles indicate that when cdAE1 is linked to the membrane, the intermonomer distance is larger at pH 5, compared to that of the purified cdAE1 segments, and exhibits a different pH-dependent behavior. An increase in the distance was also observed on binding of a metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, to cdAE1. These data provide the first demonstration of a defined change in the molecular structure of cdAE1, and also indicate that the structure under physiological conditions is different from the crystal structure determined at low pH.
机译：人红细胞阴离子交换剂1（cdAE1）的胞质结构域是红细胞骨架以及几种代谢酶和血红蛋白的组织中心。已知该蛋白质会经历可逆的pH依赖的构象变化，其特征是固有荧光发生2倍变化，斯托克斯半径发生11 A变化。尽管分子结构的确切变化是未知的，但根据pH 4.8时蛋白质的晶体结构和定点诱变研究，Zhou和Low（19）提出了cdAE1的外围蛋白质结合（PPB）域响应于增加的碱度而移动远离二聚化域。为了验证该假设，我们应用了发光共振能量转移（LRET）来测量cdAE1二聚体中Cys（201）位点（位于PPB域中）的供体探针与受体探针之间的单体间距。发现该距离随着野生型和cdAE1突变体（C317S）重组形式的pH从5增加到10而增加。此外，红细胞内外囊泡中的LRET测量表明，当cdAE1连接至膜时，与纯化的cdAE1节段相比，在pH 5时单体间距更大，并表现出不同的pH依赖性行为。在代谢酶甘油醛-3-磷酸脱氢酶与cdAE1结合时，也观察到距离增加。这些数据首次证明了cdAE1分子结构发生了确定的变化，并且还表明了在生理条件下的结构与在低pH下测定的晶体结构不同。

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