Department of Biotechnology and Biosciences, State University of Milano-Bicocca, Milano, Italy.
Department of Biotechnology and Biosciences, State University of Milano-Bicocca, Milano, Italy.
Biochim Biophys Acta Gen Subj. 2017 Nov;1861(11 Pt A):2543-2550. doi: 10.1016/j.bbagen.2017.09.002. Epub 2017 Sep 8.
Intrinsically disordered proteins (IDPs) possess a peculiar amino acid composition that makes them very soluble. Nevertheless, they can encounter aggregation in physiological and pathological contexts. In this work, we addressed the issue of how electrostatic charges can influence aggregation propensity by using the N-terminus moiety of the measles virus phosphoprotein, PNT, as a model IDP. Taking advantage of the high sequence designability of IDPs, we have produced an array of PNT variants sharing the same hydrophobicity, but differing in net charges per residue and isoelectric points (pI). The solubility and conformational properties of these proteins were analysed through biochemical and biophysical techniques in a wide range of pH values and compared with those of the green fluorescence protein (GFP), a globular protein with lower net charge per residue, but similar hydrophobicity. Tested proteins showed a solubility minimum close to their pI, as expected, but the pH-dependent decrease of solubility was not uniform and driven by the net charge per residue of each variant. A parallel behaviour was observed also in fusion proteins between PNT variants and GFP, which minimally contributes to the solubility of chimeras. Our data suggest that the overall solubility of a protein can be dictated by protein regions endowed with higher net charge per residue and, hence, prompter to respond to pH changes. This finding could be exploited for biotechnical purposes, such as the design of solubility/aggregation tags, and in studies aimed to clarify the pathological and physiological behaviour of IDPs.
无规则蛋白质（IDPs）具有独特的氨基酸组成，使其非常易溶。然而，它们在生理和病理环境中可能会发生聚集。在这项工作中，我们利用麻疹病毒磷蛋白 PNT 的 N 端部分作为模型 IDP，研究了静电电荷如何影响聚集倾向的问题。利用 IDPs 高序列设计能力，我们产生了一系列 PNT 变体，这些变体具有相同的疏水性，但每个残基的净电荷和等电点（pI）不同。通过在广泛的 pH 值范围内使用生化和生物物理技术分析这些蛋白质的溶解度和构象特性，并将其与具有较低净电荷但相似疏水性的绿色荧光蛋白（GFP）进行比较。测试的蛋白质在接近 pI 时表现出溶解度的最小值，这是预期的，但溶解度随 pH 值下降的情况并不均匀，而是由每个变体的净电荷决定的。在 PNT 变体和 GFP 之间的融合蛋白中也观察到了类似的行为，GFP 对嵌合体的溶解度贡献最小。我们的数据表明，蛋白质的整体溶解度可以由具有更高净电荷的蛋白质区域决定，因此更容易响应 pH 值的变化。这一发现可用于生物技术目的，例如设计溶解度/聚集标签，并可用于阐明 IDPs 的病理和生理行为的研究。
