Interaction study between HCV NS5A-D2 and NS5B using 19F NMR
- 327 Downloads
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 19F-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 (KD) 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 19F NMR.
Keywords19F NMR spectroscopy 19F protein labelling HCV NS5A-D2 HCV NS5B Protein–protein interaction
The NMR facilities were funded by the Région Nord-Pas de Calais, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry and the University of Lille—Sciences and Technologies. The work was supported by French National Agency for Research Grant ANR-11-JSV8-005 and French National Agency for Research on AIDS and Viral Hepatitis Grant A02014-2. We thanks the Research Federation FRABio (Université de Lille, CNRS, FR 3688, FRABio, Biochimie Structurale et Fonctionnelle des Assemblages Biomoléculaires) for providing the scientific and technical environment conducive to achieving this work.
- Pervushin K, Riek R, Wider G, Wüthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371ADSCrossRefGoogle Scholar