Complex Formation between the Hepatitis C Virus Serine Protease and A Synthetic NS4A Cofactor Peptide
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- 作者：Elisabetta Bianchi ; Andrea Urbani ; Gabriella Biasiol ; Mirko Brunetti ; Antonello Pessi ; Raffaele De Francesco ; and Christian Steinkü ; hler
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：June 24, 1997
- 年：1997
- 卷：36
- 期：25
- 页码：7890 - 7897
- 全文大小：229K
- 年卷期：v.36,no.25(June 24, 1997)
- ISSN：1520-4995
文摘
The NS3 protein of the hepatitis C virus contains a serineprotease that, upon binding to itscofactor, NS4A, is responsible for maturational cleavages that occur inthe nonstructural region of theviral polyprotein. We have studied in vitro complex formationbetween the NS3 protease domain expressedin Escherichia coli and a synthetic peptide spanning theminimal domain of the NS4A cofactor. Complexdissociation constants in the low micromolar range were measured usingdifferent techniques such asactivity titration, fluorescence titration, and pre-equilibriumanalysis of complex formation. Cofactor bindingwas strictly dependent on the glycerol content of buffer solutions andwas not significantly influenced bysubstrate saturation of the enzyme. NS4A peptide binding to NS3was accompanied by changes in thecircular dichroism spectrum in the region between 270 and 290 nm, aswell as by an enhancement oftryptophan fluorescence. Conversely, no changes in the far UVregion of the circular dichroism spectrumwere detectable. These data are indicative of induced tertiarystructure changes and suggest that thesecondary structure content of the uncomplexed enzyme does not differsignificantly from that of theNS3-cofactor complex. Pre-equilibrium measurements of complexformation showed very low valuesfor kon, suggesting conformational transitionsto be rate limiting for the association reaction.