Laboratory of Protein Conformation and Dynamics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, United States.
Laboratory of Protein Conformation and Dynamics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, United States.
Protein Expr Purif. 2021 Nov;187:105944. doi: 10.1016/j.pep.2021.105944. Epub 2021 Jul 20.
The pre-melanosomal protein (Pmel17) is a human functional amyloid that supports melanin biosynthesis within melanocytes. This occurs in the melanosome, a membrane-bound organelle with an acidic intraluminal pH. The repeat region of Pmel17 (RPT, residues 315-444) has been previously shown to form amyloid aggregates under acidic melanosomal conditions, but not under neutral cytosolic conditions, when expressed and purified using a C-terminal hexa-histidine tag (RPT-His). Given the importance of protonation states in RPT-His aggregation, we questioned whether the histidine tag influenced the pH-dependent behavior. In this report, we generated a tagless RPT by inserting a tobacco etch virus (TEV) protease recognition sequence (ENLYGQ(G/S)) immediately upstream of a native glycine residue at position 312 in Pmel17. After purification of the fusion construct using a histidine tag, cleavage with TEV protease generated a fully native RPT (nRPT) spanning resides 312-444. We characterized the aggregation of nRPT, which formed amyloid fibrils under acidic conditions (pH ≤ 6) but not at neutral pH. Characterizing the morphologies of nRPT aggregates using transmission electron microscopy revealed a pH-dependent maturation from short, curved structures at pH 4 to paired, rod-like fibrils at pH 6. This was accompanied by a secondary structural transition from mixed random coil/β-sheet at pH 4 to canonical β-sheet at pH 6. We also show that pre-formed nRPT fibrils undergo disaggregation upon dilution into pH 7 buffer. More broadly, this strategy can be utilized to generate native amyloidogenic domains from larger proteins by utilizing intrinsic N-terminal glycine or serine residues.
前黑素小体蛋白（Pmel17）是一种人类功能性淀粉样蛋白，可在黑素细胞中支持黑色素的生物合成。这发生在黑素小体中，黑素小体是一种具有酸性腔内 pH 值的膜结合细胞器。Pmel17 的重复区（RPT，残基 315-444）先前已被证明在酸性黑素小体条件下形成淀粉样聚集物，但在使用 C 端六组氨酸标签（RPT-His）表达和纯化时，在中性胞质条件下则不会。鉴于质子化状态在 RPT-His 聚集中的重要性，我们质疑标签是否会影响 pH 依赖性行为。在本报告中，我们通过在 Pmel17 的位置 312 处的天然甘氨酸残基的上游立即插入烟草蚀纹病毒（TEV）蛋白酶识别序列（ENLYGQ（G/S）），生成了无标签的 RPT。在用组氨酸标签纯化融合构建体后，用 TEV 蛋白酶切割可产生全长的天然 RPT（nRPT），跨度为残基 312-444。我们对 nRPT 的聚集进行了表征，该聚集在酸性条件（pH≤6）下形成淀粉样纤维，但在中性 pH 值下则不形成。使用透射电子显微镜对 nRPT 聚集物的形态进行表征表明，在 pH 4 时从短的、弯曲的结构到 pH 6 时的成对的、棒状纤维发生 pH 依赖性成熟。这伴随着从 pH 4 时的混合无规卷曲/β-折叠到 pH 6 时的典型β-折叠的二级结构转变。我们还表明，预形成的 nRPT 纤维在稀释到 pH 7 缓冲液时会发生解聚。更广泛地说，这种策略可以通过利用内在的 N 端甘氨酸或丝氨酸残基从较大的蛋白质中生成天然的淀粉样结构域。
