PMID: 18213401Jan 24, 2008Paper

Circular dichroism analysis for multidomain proteins: studies of the irreversible unfolding of Hepatitis C virus helicase

Acta biochimica Polonica
Agnieszka GozdekAnna M Boguszewska-Chachulska

Abstract

The non-structural protein 3 (NS3) of Hepatitis C virus (HCV) is a bifunctional enzyme with RNA-dependent NTPase/RNA helicase and serine protease activities, and thus represents a promising target for anti-HCV therapy. These functions are performed by two distinct moieties; the N-terminal protease domain and the C-terminal helicase domain that further folds into three structural subdomains. To obtain lower molecular mass proteins suitable for nuclear magnetic resonance studies of helicase-inhibitor complexes, helicase domains 1, 2, and 1+2 devoid of a hydrophobic beta-loop were overexpressed and purified. Circular dichroism studies were carried out to confirm the secondary structure content and to determine thermodynamic parameters describing the stability of the proteins. Both thermal and GuHCl-induced unfolding experiments confirmed the multidomain organization of the helicase. The unfolding transition observed for domain 1+2 was in agreement with the model of two well-resolved successive steps corresponding to the independent unfolding of domains 1 and 2, respectively. In the case of the full-length helicase, the presence of domain 3 remarkably changed the transition profile, leading to fast and irreversible transformation o...Continue Reading

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