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PDBsum entry 1alg
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Oxidoreductase
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PDB id
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1alg
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References listed in PDB file
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Key reference
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Title
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Denaturation and reactivation of dimeric human glutathione reductase--An assay for folding inhibitors.
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Authors
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A.Nordhoff,
C.Tziatzios,
J.A.Van den broek,
M.K.Schott,
H.R.Kalbitzer,
K.Becker,
D.Schubert,
R.H.Schirmer.
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Ref.
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Eur J Biochem, 1997,
245,
273-282.
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PubMed id
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Abstract
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Human glutathione reductase (GR; which catalyzes the reaction NADPH + GSSG + H+
--> 2 GSH + NADP+) is an obligatory FAD-containing homodimer of known
geometry. Native human GR, a potential target of antimalarial and cytostatic
agents, cannot be dissociated by dilution or by means of subunit-interface
mimetics, similarly to well-studied viral dimeric proteins. However, ab initio
folding and/or dimerization of human GR can be inhibited by point mutations or
by peptides corresponding to subunit-interface areas, for example synthetic
peptide P11, which represents the intersubunit-contact helix H11. The structure
of this peptide, which might assist inhibitor design, was solved by
high-resolution NMR spectroscopy. Residues 440-453, were found to be alpha
helical in the isolated peptide. To quantitate the efficacy of inhibitors such
as P11, we developed the following unfolding/reactivation assay. The effects of
various guanidine hydrochloride (Gdn/HCl) concentrations were studied by
analytical ultracentrifugation. It was shown that human GR denatured by greater
than 3 M Gdn/HCl is monomeric and free of FAD. Circular-dichroism experiments at
223 nm indicated a half-life of approximately 20 s at 20 degrees C for the
unfolding process. To optimize the reactivation yield, four parameters [protein
concentration (x) in the range 0.3-10 microg/ml, cofactor supplementation,
temperature (y: 0-32 degrees C), and time (0-72 h)] were varied systematically,
and a reactivation score z was given to each constellation of parameters. This
type of analysis might be useful to optimize refolding and activation yields for
other proteins. For human GR, the highest recovery was found not to occur at one
of the corners of the x,y plane, but close to its center. Consequently, the
optimal assay conditions for folding and dimerization inhibitors are as follows.
The enzyme (at 300 microg/ml) is denatured by 5 M guanidine hydrochloride/5 mM
dithiothreitol, then reactivated by dilution to 1 microg/ml at pH 6.9 and 20
degrees C. In the absence of inhibitors, this procedure leads to 70% of the
control activity within 8 h. Peptides representing the upper subunit interface
(for instance residues 436-478) of human GR were found to inhibit refolding with
EC50% values in the micromolar range, whereas fragments from other regions of
the protein had no influence on this process. For peptide P11, the EC50% value
was 20 microM. In conclusion, hGR, enzyme with a tight intersubunit contact area
of 21 nm2, appears to be suitable for studying protein folding, dimerization,
and prosthetic-group complexation in the absence and presence of compounds that
inhibit these processes. There is a shortage, at least for oligomeric enzymes of
eukaryotes, of published systematic studies on protein (re)activation.
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