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A comparative analysis of the fluorescence properties of the wild-type and active site mutants of the hepatitis C virus autoprotease NS2-3.

Foster TL, Tedbury PR, Pearson AR, Harris M - Biochim. Biophys. Acta (2009)

Bottom Line: The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage.We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage.Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

ABSTRACT
Hepatitis C virus encodes an autoprotease, NS2-3, which is required for processing of the viral polyprotein between the non-structural NS2 and NS3 proteins. This protease activity is vital for the replication and assembly of the virus and therefore represents a target for the development of anti-viral drugs. The mechanism of this auto-processing reaction is not yet clear but the protease activity has been shown to map to the C-terminal region of NS2 and the N-terminal serine protease region of NS3. The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage. Here, intrinsic tryptophan fluorescence has been used to assess refolding in the wild-type protein and specific active site mutants. We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage. Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.

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Related in: MedlinePlus

Fluorescence emission spectra of wild-type and mutant NS2-3 precursor. (A) Comparison of the fluorescence emission spectra of WT NS2-3 at 4 °C or 25 °C under denaturing (solid line), and refolded (dashed line), conditions. Low temperature inhibits activity but allows folding of the NS2-3 precursor. The vertical line indicates the tryptophan emission maximum of NS2-3. (B) Inhibition of activity by zinc chelation is directly linked to the lack of refolding in the presence of 10 mM EDTA. (C) Fluorescence emission spectra of the active site mutants H956A and C997A.
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fig4: Fluorescence emission spectra of wild-type and mutant NS2-3 precursor. (A) Comparison of the fluorescence emission spectra of WT NS2-3 at 4 °C or 25 °C under denaturing (solid line), and refolded (dashed line), conditions. Low temperature inhibits activity but allows folding of the NS2-3 precursor. The vertical line indicates the tryptophan emission maximum of NS2-3. (B) Inhibition of activity by zinc chelation is directly linked to the lack of refolding in the presence of 10 mM EDTA. (C) Fluorescence emission spectra of the active site mutants H956A and C997A.

Mentions: Direct comparison of any differences in the folding properties of the WT protease and the mutants requires analysis of WT NS2-3 in a pre-cleavage state. The auto-cleavage of JFH-1 NS2-3 protease, unlike J4 NS2-3, does not proceed at 4 °C (Fig. 2F); therefore, this made it possible to assess and compare the folding of WT and mutant NS2-3 pre-cleavage. To investigate whether refolding at low temperature had any differential effect on the conformation of the protease compared to that at 25 °C, we measured the fluorescence emission spectra of denatured and refolded WT NS2-3 precursor at 25 °C and at 4 °C after excitation at 295 nm. The emission spectra of denatured WT NS2-3 at the two temperatures show similar emission maxima (373 nm at 25 °C, 371 nm at 4 °C) and intensities demonstrating that the polarity of the microenvironment around the tryptophan residues of NS2-3 at the two temperatures is similar (Fig. 4A). Upon refolding overnight at 4 °C or 25 °C, wild-type NS2-3 exhibited both a decrease in the tryptophan fluorescence intensity and a blue shift of the maximal emission wavelength to 342 nm, indicating that the environment around the tryptophan residues had become more hydrophobic (Fig. 4A). The folding of the NS2-3 precursor therefore occurs at 4 °C; this temperature represents a suitable condition under which to study the conformation of the precursor state of the wild-type enzyme. Comparisons of the fluorescence properties of the WT protease with those of the mutants at 4 °C were therefore assessed and detailed in section 3.3.


A comparative analysis of the fluorescence properties of the wild-type and active site mutants of the hepatitis C virus autoprotease NS2-3.

Foster TL, Tedbury PR, Pearson AR, Harris M - Biochim. Biophys. Acta (2009)

Fluorescence emission spectra of wild-type and mutant NS2-3 precursor. (A) Comparison of the fluorescence emission spectra of WT NS2-3 at 4 °C or 25 °C under denaturing (solid line), and refolded (dashed line), conditions. Low temperature inhibits activity but allows folding of the NS2-3 precursor. The vertical line indicates the tryptophan emission maximum of NS2-3. (B) Inhibition of activity by zinc chelation is directly linked to the lack of refolding in the presence of 10 mM EDTA. (C) Fluorescence emission spectra of the active site mutants H956A and C997A.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2824112&req=5

fig4: Fluorescence emission spectra of wild-type and mutant NS2-3 precursor. (A) Comparison of the fluorescence emission spectra of WT NS2-3 at 4 °C or 25 °C under denaturing (solid line), and refolded (dashed line), conditions. Low temperature inhibits activity but allows folding of the NS2-3 precursor. The vertical line indicates the tryptophan emission maximum of NS2-3. (B) Inhibition of activity by zinc chelation is directly linked to the lack of refolding in the presence of 10 mM EDTA. (C) Fluorescence emission spectra of the active site mutants H956A and C997A.
Mentions: Direct comparison of any differences in the folding properties of the WT protease and the mutants requires analysis of WT NS2-3 in a pre-cleavage state. The auto-cleavage of JFH-1 NS2-3 protease, unlike J4 NS2-3, does not proceed at 4 °C (Fig. 2F); therefore, this made it possible to assess and compare the folding of WT and mutant NS2-3 pre-cleavage. To investigate whether refolding at low temperature had any differential effect on the conformation of the protease compared to that at 25 °C, we measured the fluorescence emission spectra of denatured and refolded WT NS2-3 precursor at 25 °C and at 4 °C after excitation at 295 nm. The emission spectra of denatured WT NS2-3 at the two temperatures show similar emission maxima (373 nm at 25 °C, 371 nm at 4 °C) and intensities demonstrating that the polarity of the microenvironment around the tryptophan residues of NS2-3 at the two temperatures is similar (Fig. 4A). Upon refolding overnight at 4 °C or 25 °C, wild-type NS2-3 exhibited both a decrease in the tryptophan fluorescence intensity and a blue shift of the maximal emission wavelength to 342 nm, indicating that the environment around the tryptophan residues had become more hydrophobic (Fig. 4A). The folding of the NS2-3 precursor therefore occurs at 4 °C; this temperature represents a suitable condition under which to study the conformation of the precursor state of the wild-type enzyme. Comparisons of the fluorescence properties of the WT protease with those of the mutants at 4 °C were therefore assessed and detailed in section 3.3.

Bottom Line: The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage.We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage.Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

ABSTRACT
Hepatitis C virus encodes an autoprotease, NS2-3, which is required for processing of the viral polyprotein between the non-structural NS2 and NS3 proteins. This protease activity is vital for the replication and assembly of the virus and therefore represents a target for the development of anti-viral drugs. The mechanism of this auto-processing reaction is not yet clear but the protease activity has been shown to map to the C-terminal region of NS2 and the N-terminal serine protease region of NS3. The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage. Here, intrinsic tryptophan fluorescence has been used to assess refolding in the wild-type protein and specific active site mutants. We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage. Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.

Show MeSH
Related in: MedlinePlus