Analytical and BioNMR Laboratory, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1/a, Budapest, 1117, Hungary.
Department of Biochemistry, Eötvös Loránd University, Pázmány Péter sétány 1/c, Budapest, 1117, Hungary.
Biomol NMR Assign. 2023 Dec;17(2):309-314. doi: 10.1007/s12104-023-10160-4. Epub 2023 Oct 20.
Protein p53 is mostly known for playing a key role in tumour suppression, and mutations in the p53 gene are amongst the most frequent genomic events accompanying oncogenic transformation. Continuous research is conducted to target disordered proteins/protein regions for cancer therapy, for which atomic level information is also necessary. The disordered N-terminal part of p53 contains the transactivation and the proline-rich domains-which besides being abundant in proline residues-contains repetitive Pro-Ala motifs. NMR assignment of such repetitive, proline-rich regions is challenging due to the lack of amide protons in the H-detected approaches, as well as due to the small chemical shift dispersion. In the present study we perform the full assignment of the p53 region by applying a combination of H- and H-detected NMR experiments. We also show the increased information content when using real-time homo- and heteronuclear decoupled acquisition schemes. On the other hand, we highlight the presence of minor proline species, and using Pro-selective experiments we determine the corresponding cis or trans conformation. Secondary chemical shifts for (C-C) atoms indicate the disordered nature of this region, with expected helical tendency for the TAD1 region. As the role of the proline-rich domain is yet not well understood our results can contribute to further successful investigations.
蛋白质 p53 主要以在肿瘤抑制中发挥关键作用而闻名，而 p53 基因的突变是伴随致癌转化的最常见的基因组事件之一。为了进行癌症治疗，人们一直在持续研究靶向无序蛋白/蛋白区域，这也需要原子水平的信息。p53 的无序 N 端部分包含转录激活和脯氨酸丰富区——除了富含脯氨酸残基外，还包含重复的脯氨酸-丙氨酸基序。由于 H 检测方法中缺乏酰胺质子，以及化学位移分散较小，因此对这种重复的脯氨酸丰富区域进行 NMR 分配是具有挑战性的。在本研究中，我们通过应用 H 和 H 检测 NMR 实验的组合来完成 p53 区域的完整分配。我们还展示了使用实时同核和异核去耦采集方案时增加的信息量。另一方面，我们强调了存在少量的脯氨酸物质，并使用脯氨酸选择性实验确定了相应的顺式或反式构象。（C-C）原子的二级化学位移表明该区域具有无序性质，TAD1 区域具有预期的螺旋倾向。由于脯氨酸丰富区的作用尚未得到很好的理解，我们的结果可以为进一步的成功研究做出贡献。
