Complex formation between the hepatitis C virus serine protease and a synthetic NS4A cofactor peptide

Elisatta Bianchi, Andrea Urbani, Gabriella Biasiol, Mirko Brunetti, Antonello Pessi, Raffaele De Francesco, Christian Steinkühler

Research output: Contribution to journalArticlepeer-review


The NS3 protein of the hepatitis C virus contains a serine protease that, upon binding to its cofactor, NS4A, is responsible for maturational cleavages that occur in the nonstructural region of the viral polyprotein. We have studied in vitro complex formation between the NS3 protease domain expressed in Escherichia coli and a synthetic peptide spanning the minimal domain of the NS4A cofactor. Complex dissociation constants in the low micromolar range were measured using different techniques such as activity titration, fluorescence titration, and pre-equilibrium analysis of complex formation. Cofactor binding was strictly dependent on the glycerol content of buffer solutions and was not significantly influenced by substrate saturation of the enzyme. NS4A peptide binding to NS3 was accompanied by changes in the circular dichroism spectrum in the region between 270 and 290 nm, as well as by an enhancement of tryptophan fluorescence. Conversely, no changes in the far UV region of the circular dichroism spectrum were detectable. These data are indicative of induced tertiary structure changes and suggest that the secondary structure content of the uncomplexed enzyme does not differ significantly from that of the NS3-cofactor complex. Pre-equilibrium measurements of complex formation showed very low values for k(on), suggesting conformational transitions to be rate limiting for the association reaction.

Original languageEnglish
Pages (from-to)7890-7897
Number of pages8
Issue number25
Publication statusPublished - Jun 24 1997

ASJC Scopus subject areas

  • Biochemistry


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