A comparative analysis of the fluorescence properties of the wild-type and active site mutants of the hepatitis C virus autoprotease NS2-3.
Hepatitis C virus encodes an autoprotease, NS2-3, which is required for processing of the viral polyprotein between the non-structural NS2 and NS3 proteins. This protease activity is vital for the replication and assembly of the virus and therefore represents a target for the development of anti-viral drugs. The mechanism of this auto-processing reaction is not yet clear but the protease activity has been shown to map to the C-terminal region of NS2 and the N-terminal serine protease region of NS3. The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage. Here, intrinsic tryptophan fluorescence has been used to assess refolding in the wild-type protein and specific active site mutants. We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage. Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.
Foster
Toshana L
TL
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Tedbury
Philip R
PR
Pearson
Arwen R
AR
Harris
Mark
M
D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics*; D008958:Models, Molecular; D017354:Point Mutation; D017510:Protein Folding; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics*
D014764:Viral Proteins; D014364:Tryptophan; D003546:Cysteine Endopeptidases; C107824:NS2-3 protease