Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET-Universidad de Buenos Aires, Buenos Aires, Argentina.
PLoS One. 2018 Aug 23;13(8):e0202808. doi: 10.1371/journal.pone.0202808. eCollection 2018.
Plants in arid zones are constantly exposed to drought stress. The ASR protein family (Abscisic, Stress, Ripening) -a subgroup of the late embryogenesis abundant superfamily- is involved in the water stress response and adaptation to dry environments. Tomato ASR1, as well as other members of this family, is an intrinsically disordered protein (IDP) that functions as a transcription factor and a chaperone. Here we employed different biophysical techniques to perform a deep in vitro characterization of ASR1 as an IDP and showed how both environmental factors and in vivo targets modulate its folding. We report that ASR1 adopts different conformations such as α-helix or polyproline type II in response to environmental changes. Low temperatures and low pH promote the polyproline type II conformation (PII). While NaCl increases PII content and slightly destabilizes α-helix conformation, PEG and glycerol have an important stabilizing effect of α-helix conformation. The binding of Zn2+in the low micromolar range promotes α-helix folding, while extra Zn2+ results in homo-dimerization. The ASR1-DNA binding is sequence specific and dependent on Zn2+. ASR1 chaperone activity does not change upon the structure induction triggered by the addition of Zn2+. Furthermore, trehalose, which has no effect on the ASR1 structure by itself, showed a synergistic effect on the ASR1-driven heat shock protection towards the reporter enzyme citrate synthase (CS). These observations prompted the development of a FRET reporter to sense ASR1 folding in vivo. Its performance was confirmed in Escherichia coli under saline and osmotic stress conditions, representing a promising probe to be used in plant cells. Overall, this work supports the notion that ASR1 plasticity is a key feature that facilitates its response to drought stress and its interaction with specific targets.
干旱区的植物经常受到干旱胁迫的影响。ASR 蛋白家族（脱落酸、胁迫、成熟）——晚期胚胎丰富超家族的一个亚组——参与了水胁迫反应和对干燥环境的适应。番茄 ASR1 以及该家族的其他成员，是一种无序蛋白（IDP），作为转录因子和伴侣发挥作用。在这里，我们采用了不同的生物物理技术对 ASR1 作为 IDP 进行了深入的体外特性分析，并展示了环境因素和体内靶标如何调节其折叠。我们报告说，ASR1 会根据环境变化采用不同的构象，如α-螺旋或聚脯氨酸 II 型。低温和低 pH 促进聚脯氨酸 II 型构象（PII）。虽然 NaCl 增加了 PII 含量并略微降低了α-螺旋构象的稳定性，但 PEG 和甘油对α-螺旋构象具有重要的稳定作用。在低微摩尔范围内结合 Zn2+会促进α-螺旋折叠，而额外的 Zn2+会导致同二聚化。ASR1 与 DNA 的结合是序列特异性的，并依赖于 Zn2+。Zn2+触发的结构诱导不会改变 ASR1 的伴侣活性。此外，海藻糖本身对 ASR1 结构没有影响，但对 ASR1 驱动的热休克保护柠檬酸合酶（CS）具有协同作用。这些观察结果促使我们开发了一种 FRET 报告蛋白来检测体内 ASR1 的折叠。在大肠杆菌中，在盐和渗透胁迫条件下，其性能得到了验证，这代表了一种在植物细胞中很有前途的探针。总的来说，这项工作支持了 ASR1 可塑性是促进其对干旱胁迫反应及其与特定靶标相互作用的关键特征的观点。
