Protein Engineering, Vol. 14, No. 8, 573-582,
August 2001
© 2001 Oxford University Press
Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies
1 These two authors contributed equally to this work. Department of Structural Chemistry, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
RNA helicases represent a family of enzymes that unwind double-stranded (ds) RNA in a nucleoside triphosphate (NTP)-dependent fashion and which are required in all aspects of cellular RNA metabolism and processing. The hepatitis C virus (HCV) non-structural 3 (NS3) protein possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion of the 631 amino acid residue bifunctional enzyme. The HCV NS3 RNA helicase is of key importance in the life cycle of HCV, which makes it a target for the development of therapeutics. However, neither the precise mechanism nor the substrate structure has been defined for this enzyme. For nuclear magnetic resonance (NMR)-based drug discovery methods and for mechanistic studies we engineered, prepared and characterized various truncated constructs of the 451-residue HCV NS3 RNA helicase. Our goal was to produce smaller fragments of the enzyme, which would be amenable to solution NMR techniques while retaining their native NTP and/or nucleic acid binding sites. Solution conditions were optimized to obtain high-quality heteronuclear NMR spectra of nitrogen-15 isotope-labeled constructs, which are typical of well-folded monomeric proteins. Moreover, NMR binding studies and functional data directly support the correct folding of these fragments.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  D. Liu, W. T. Windsor, and D. F. Wyss Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2 Protein Sci., December 1, 2003; 12(12): 2757 - 2767. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. I. Lam, D. Keeney, and D. N. Frick Two Novel Conserved Motifs in the Hepatitis C Virus NS3 Protein Critical for Helicase Action J. Biol. Chem., November 7, 2003; 278(45): 44514 - 44524. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Daujotyte, G. Vilkaitis, L. Manelyte, J. Skalicky, T. Szyperski, and S. Klimasauskas Solubility engineering of the HhaI methyltransferase Protein Eng. Des. Sel., April 1, 2003; 16(4): 295 - 301. [Abstract] [Full Text] [PDF]
|
|