Spectrin, the major protein of the erythrocyte membrane skeleton has canonically been thought to only serve a structural function. We have previously described a novel chaperone‐like property of spectrin and also hypothesized that the chaperone activity and binding of a hydrophobic ligand, Prodan are localized in the self‐association domain. Here we probe the location and molecular origin of the chaperone activity of multi‐domain spectrin using a selection of individual recombinant spectrin domains, which we have characterized using intrinsic tryptophan fluorescence and CD spectroscopy to show their identity to native spectrin. Aggregation assays using insulin, ADH, α‐ and β‐globin as well as enzyme refolding assays using alkaline phosphatase and α‐glucosidase show that the chaperone activity is not only localized in the self‐association domain but is a generalized property of spectrin domains. This is to our understanding, a unique feature in the case of modular multi‐repeat proteins, possibly implicating that the large family of “spectrin‐repeat” domain containing proteins may also have chaperone like property. Substrate selectivity of chaperone activity as evidenced by the preferential protection of α‐ over β‐globin chains is seen; which has implications in hemoglobin diseases. Moreover, enzyme‐refolding assays also indicate alternate modes of chaperone action. We propose that the molecular origin of chaperone activity resides in the surface exposed hydrophobic patches of the spectrin domains as shown by ANS (1‐anilinonaphthalene‐8‐sulfonic acid) and Prodan (6‐propionyl‐2[dimethylamino]‐naphthalene) binding. We also show that Prodan does indeed have a unique binding site on spectrin located at the self‐association domain.

中文翻译：

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本地化红系血影蛋白的伴侣活性。

血影蛋白是红细胞膜骨架的主要蛋白质，通常被认为仅起结构作用。我们先前已经描述了血影蛋白的新型伴侣状性质，并且还假设伴侣活性和疏水性配体Prodan的结合位于自缔合域中。在这里，我们通过选择单个重组血影蛋白域来探测多域血影蛋白伴侣分子活性的位置和分子起源，我们已使用固有色氨酸荧光和CD光谱对其进行了表征，以显示它们与天然血影蛋白的身份。使用胰岛素，ADH，α-和β-珠蛋白以及使用碱性磷酸酶和α-葡萄糖苷酶的酶复性分析表明，伴侣蛋白活性不仅位于自缔合域，而且是血影蛋白域的普遍性质。据我们了解，这是模块化多重复蛋白的独特特征，可能暗示包含“蛋白-重复”结构域的蛋白质家族也可能具有类似伴侣的特性。观察到伴侣分子活性的底物选择性，如α-优于β-珠蛋白链所证明的；对血红蛋白疾病有影响。此外，酶复性测定还表明了伴侣作用的替代模式。我们建议伴侣活性的分子起源在于血影蛋白域的表面暴露疏水斑块，如ANS（1-苯胺基萘-8-磺酸）和Prodan（6-丙酰基-2 [二甲基氨基]-萘）结合所示。我们还表明，Prodan确实在位于自缔合域的血影蛋白上具有独特的结合位点。