title: Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro.
abstract: HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
authors.forename: Bartek
authors.initials: B
authors.lastname: Sikora
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: Chen
authors.forename: Cheryl F
authors.initials: CF
authors.lastname: Lichti
authors.forename: Melody K
authors.initials: MK
authors.lastname: Harrison
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Jennings
authors.forename: Yong
authors.initials: Y
authors.lastname: Tang
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Tackett
authors.forename: John B
authors.initials: JB
authors.lastname: Jordan
authors.forename: Joshua
authors.initials: J
authors.lastname: Sakon
authors.forename: Craig E
authors.initials: CE
authors.lastname: Cameron
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Raney
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D002384:Catalysis; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry*; D004265:DNA Helicases / Q000737:chemistry; D016174:Hepacivirus / Q000201:enzymology*; D066298:In Vitro Techniques; D007700:Kinetics; D008027:Light; D009691:Nucleic Acid Denaturation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000251:Adenosine Triphosphatases; D003432:Cross-Linking Reagents; D004247:DNA; D004265:DNA Helicases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases