Dujardin Marie, Cantrelle François-Xavier, Lippens Guy, Hanoulle Xavier
Université de Lille, CNRS, UMR 8576 - UGSF, 59000, Lille, France.
LISBP, Université de Toulouse, CNRS, INRA, INSA, UPS 135 avenue de Rangueil, 31077, Toulouse, France.
J Biomol NMR. 2018 Jan;70(1):67-76. doi: 10.1007/s10858-017-0159-9. Epub 2017 Dec 7.
The non structural protein 5A (NS5A) regulates the replication of the hepatitis C viral RNA through a direct molecular interaction of its domain 2 (NS5A-D2) with the RNA dependent RNA polymerase NS5B. Because of conflicting data in the literature, we study here this molecular interaction using fluorinated versions of the NS5A-D2 protein derived from the JFH1 Hepatitis C Virus strain. Two methods to prepare fluorine-labelled NS5A-D2 involving the biosynthetic incorporation of a F-tryptophan using 5-fluoroindole and the posttranslational introduction of fluorine by chemical conjugation of 2-iodo-N-(trifluoromethyl)acetamide with the NS5A-D2 cysteine side chains are presented. The dissociation constants (K) between NS5A-D2 and NS5B obtained with these two methods are in good agreement, and yield values comparable to those derived previously from a surface plasmon resonance study. We compare benefits and limitations of both labeling methods to study the interaction between an intrinsically disordered protein and a large molecular target by F NMR.
非结构蛋白5A（NS5A）通过其结构域2（NS5A-D2）与RNA依赖性RNA聚合酶NS5B的直接分子相互作用来调节丙型肝炎病毒RNA的复制。由于文献中的数据相互矛盾，我们在此使用源自JFH1丙型肝炎病毒株的NS5A-D2蛋白的氟化版本来研究这种分子相互作用。本文介绍了两种制备氟标记的NS5A-D2的方法，一种是使用5-氟吲哚通过生物合成掺入F-色氨酸，另一种是通过将2-碘-N-（三氟甲基）乙酰胺与NS5A-D2半胱氨酸侧链进行化学偶联来进行氟的翻译后引入。用这两种方法获得的NS5A-D2与NS5B之间的解离常数（K）吻合良好，所得值与先前表面等离子体共振研究得出的值相当。我们比较了两种标记方法的优缺点，以便通过F NMR研究内在无序蛋白与大分子靶标之间的相互作用。
