Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, D-06120 Halle/Saale, Germany.
J Med Chem. 2013 Jun 13;56(11):4252-63. doi: 10.1021/jm400446b. Epub 2013 May 17.
Chemical cross-linking combined with an enzymatic digestion and mass spectrometric analysis of the reaction products has evolved into an alternative strategy to structurally resolve protein complexes. We investigated conformational changes in peroxisome proliferator-activated receptor α (PPARα) upon ligand binding. Using E. coli cells with a special tRNA/aminoacyl-tRNA synthetase pair, two PPARα variants were prepared in which Leu-258 or Phe-273 were site-specifically replaced by the genetically encoded photoreactive amino acid p-benzoylphenylalanine (Bpa). PPARα variants were subjected to UV-induced cross-linking, both in the absence and in the presence of ligands. After the photo-cross-linking reaction, reaction mixtures were enzymatically digested and peptides were analyzed by mass spectrometry. The inter-residue distances disclosed by the photochemical cross-links served to monitor conformational changes in PPARα upon agonist and antagonist binding. The data obtained with our strategy emphasize the potential of genetically encoded internal photo-cross-linkers in combination with mass spectrometry as an alternative method to monitor in-solution 3D-protein structures.
化学交联与酶解及反应产物的质谱分析相结合，已经发展成为一种结构解析蛋白质复合物的替代策略。我们研究了配体结合后过氧化物酶体增殖物激活受体α（PPARα）的构象变化。利用带有特殊 tRNA/氨酰-tRNA 合成酶对的大肠杆菌细胞，我们制备了两个 PPARα 变体，其中 Leu-258 或 Phe-273 被遗传编码的光反应性氨基酸对苯甲酰基苯丙氨酸（Bpa）定点取代。将 PPARα 变体在有无配体的情况下进行 UV 诱导交联，然后进行光交联反应。反应混合物进行酶解，肽段通过质谱进行分析。光化学交联揭示的残基间距离用于监测激动剂和拮抗剂结合时 PPARα 的构象变化。我们的策略获得的数据强调了遗传编码内部光交联剂与质谱相结合作为监测溶液中 3D 蛋白质结构的替代方法的潜力。
