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

• Autorzy: Gozdek A. , Stankiewicz-Drogon A. , Poznanski J. , Boguszewska-Chachulska A.M.
• Języki publikacji: EN

Abstrakty

EN

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 β-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 of partially unfolded protein.

Słowa kluczowe

EN

helicase; non-structural protein; protein; protein unfolding; Lumry-Eyring model; circular dichroism; hepatitis C virus; thermal resistance

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2008
• Tom: 55
• Numer: 1
• Opis fizyczny: p.57-66,fig.,ref.

Twórcy

autor

Gozdek A.

• Polish Academy of Sciences, A.Pawinskiego 5a, 02-106 Warsaw, Poland

autor

Stankiewicz-Drogon A.

autor

Poznanski J.

autor

Boguszewska-Chachulska A.M.

Bibliografia

• Boguszewska-Chachulska AM, Krawczyk M, Stankiewicz A, Gozdek A, Haenni A.-L, Strokovskaya L (2004) Direct fluorometric measurement of hepatitis C virus helicase activity. FEBS Lett 567: 253-258.
• Boguszewska-Chachulska AM, Krawczyk M, Najda A, Kopańska A, Stankiewicz A, Zagorski-Ostoja W, Bretner M (2006) Searching for a new anti-HCV therapy: synthesis and properties of tropolone derivatives. Biochem Biophys Res Commun 341: 641-647.
• Bohm G, Muhr R, Jaenicke R (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng 5: 191-195.
• Bretner M (2005) Existing and future therapeutic options for hepatitis C virus infection. Acta Biochim Polon 52: 57-70.
• Cieplak M, Sulkowska JI (2005) Thermal unfolding of proteins. J Chem Phys 123: 194-908.
• Frost AA, Pearson RG (1961) Transition-state theory: comparison of collision and transition state theories. In Kinetics and Mechanism. Frost AA, Pearson RG, eds, pp 93-99. 2nd ed. John Wiley and Sons, New York, USA.
• Gesell JJ, Liu D, Madison VS, Hesson T, Wang YS, Weber PC, Wyss DF (2001) Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies. Protein Eng 14: 573-582.
• Gordon CP, Keller PA (2005) Control of hepatitis C: a medicinal chemistry perspective. J Med Chem 48: 1-20.
• Hadziyannis SJ Jr, Sette H, Morgan TR, Balan V, Diago M, Marcellin P, Ramadori G, Jr Bodenheimer H, Bernstein D, Rizzetto M, Zeuzem S, Pockros PJ, Lin A, Ackrill AM, PEGASYS International Study Group (2004) Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Ann Intern Med 140: 346-355.
• Kelly SM, Price NC (1997) The application of circular dichroism to studies of protein folding and unfolding. Biochem Biophys Acta 1338: 161-185.
• Kim DW, Gwack Y, Han JH, Choe J (1995) C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem Biophys Res Commun 215: 160-166.
• Kwong AD, Kim JL, Lin C (2000) Structure and function of hepatitis C virus NS3 helicase. Curr Top Microbiol Immunol 242: 171-196.
• Lam AM, Keeney D, Frick DN (2003) Two novel conserved motifs in the hepatitis C virus NS3 protein critical for helicase action. J Biol Chem 278: 44514^4524.
• Levin MK, Patel SS (2002) Helicase from hepatitis C virus, energetics of DNA binding. J Biol Chem 277: 29377- 29385.
• Liu D, Wyss DF (2000) Sequence-specific 1H, 15N and 13C resonance assignments for an engineered arginine-rich domain of the hepatitis C virus NS3 RNA helicase. J Biomol NMR 18: 279-280.
• Liu D, Wyss DF (2001) Backbone 1H, 15N and 13C resonance assignments of the NTPase subdomain of the hepatitis C virus NS3 RNA helicase. J Biomol NMR 19: 283-284.
• Liu D, Windsor WT, Wyss DF (2003) Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2. Protein Sci 12: 2757-2767.
• Lumry R, Eyring H (1954) Conformation changes of proteins. J Phys Chem 58: 110-120.
• Marquardt DW (1963) An algorithm for least-squares estimation of non-linear parameters. J Soc Ind App Math 11: 431-141.
• Rosenberg S (2001) Recent advances in the molecular biology of hepatitis C virus. J Mol Biol 313: 451-464.
• Sarver RW, Rogers JM, Stockman BJ, Epps DE, DeZwaan J, Harris MS, Baldwin ET (2002) Physical methods to determine the binding mode of putative ligands for hepatitis C virus NS3 helicase. Anal Biochem 309: 186-195.
• Williams T, Kelley C (2007) Gnuplot version 4.1, Copyright (C) 1986-2007.
• Yao N, Hesson T, Cable M, Hong Z, Kwong AD, Le HV, Weber PC (1997) Structure of the hepatitis C virus RNA helicase domain. Nat Struct Mol Biol 6: 463-467.
• Zagulski M, Kressler D, Becam AM, Rytka J, Herbert CJ (2003) Mak5p, which is required for the maintenance of the Ml dsRNA virus, is encoded by the yeast ORF YBR142w and is involved in the biogenesis of the 60S subunit of the ribosome. Mol Genet Genomics 270: 216- 224.