Core Protein—Nucleic Acid Interactions in Hepatitis C Virus as Revealed by Raman and Circular Dichroism Spectroscopy

To serve as models of protein–nucleic acid complexes, annealed complexes of several sequential lysine–glycine polypeptides with DNA have been formed. Circular dichroic spectra of the DNA contained in the complexes indicates that several discrete conformations of DNA can exist when complexed.

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

Acta Biochimica Polonica ◽

10.18388/abp.2008_3201 ◽

2008 ◽

Vol 55 (1) ◽

pp. 57-66 ◽

Cited By ~ 6

Author(s):

Agnieszka Gozdek ◽

Anna Stankiewicz-Drogoń ◽

Jarosław Poznański ◽

Anna M Boguszewska-Chachulska

Keyword(s):

Circular Dichroism ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Helicase Domain ◽

Structural Protein ◽

Promising Target ◽

Domain 3 ◽

Partially Unfolded ◽

The Stability ◽

Circular Dichroïsm

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 of partially unfolded protein.

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Right-handed triplex formed between peptide nucleic acid PNA-T8 and poly(dA) shown by linear and circular dichroism spectroscopy

Journal of the American Chemical Society ◽

10.1021/ja00068a001 ◽

1993 ◽

Vol 115 (15) ◽

pp. 6477-6481 ◽

Cited By ~ 90

Author(s):

Seog K. Kim ◽

Peter E. Nielsen ◽

Michael Egholm ◽

Ole Buchardt ◽

Rolf H. Berg ◽

...

Keyword(s):

Circular Dichroism ◽

Nucleic Acid ◽

Peptide Nucleic Acid ◽

Circular Dichroism Spectroscopy ◽

Circular Dichroïsm

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Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124215 ◽

1992 ◽

Vol 50 (1) ◽

pp. 762-763

Author(s):

Stephen D. Jett

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Nucleic Acid Binding ◽

Mobility Shift ◽

Carbon Coated ◽

Nucleoprotein Complexes ◽

Mobility Shift Assay ◽

Protein Nucleic Acid ◽

Gel Mobility Shift ◽

Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

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