Centre de Biochimie Structurale (CBS), INSERM, CNRS, Université de Montpellier, 29 rue de Navacelles, 34090, Montpellier, France.
Laboratoire de Biologie Cellulaire et Moléculaire, (LBCM-EA4558 Vaccination Antiparasitaire), UFR Pharmacie, Université de Montpellier, Montpellier, France.
Angew Chem Int Ed Engl. 2018 Mar 26;57(14):3598-3601. doi: 10.1002/anie.201711530. Epub 2018 Mar 7.
Homorepeat (HR) proteins are involved in key biological processes and multiple pathologies, however their high-resolution characterization has been impaired due to their homotypic nature. To overcome this problem, we have developed a strategy to isotopically label individual glutamines within HRs by combining nonsense suppression and cell-free expression. Our method has enabled the NMR investigation of huntingtin exon1 with a 16-residue polyglutamine (poly-Q) tract, and the results indicate the presence of an N-terminal α-helix at near neutral pH that vanishes towards the end of the HR. The generality of the strategy was demonstrated by introducing a labeled glutamine into a pathological version of huntingtin with 46 glutamines. This methodology paves the way to decipher the structural and dynamic perturbations induced by HR extensions in poly-Q-related diseases. Our approach can be extended to other amino acids to investigate biological processes involving proteins containing low-complexity regions (LCRs).
同源重复（HR）蛋白参与关键的生物过程和多种病理，但由于其同型性质，其高分辨率特征一直受到阻碍。为了克服这个问题，我们开发了一种通过无义抑制和无细胞表达相结合在 HR 中对单个谷氨酰胺进行同位素标记的策略。我们的方法使能够对具有 16 个残基聚谷氨酰胺（poly-Q）片段的 huntingtin exon1 进行 NMR 研究，结果表明在接近中性 pH 值时存在一个 N 端α-螺旋，该螺旋在 HR 末端消失。通过在含有 46 个谷氨酰胺的 huntingtin 的病理版本中引入标记的谷氨酰胺，证明了该策略的通用性。该方法为破译与 poly-Q 相关疾病中 HR 延伸引起的结构和动力学扰动铺平了道路。我们的方法可以扩展到其他氨基酸，以研究涉及含有低复杂度区域（LCR）的蛋白质的生物学过程。
