1alg
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(New page: 200px<br /> <applet load="1alg" size="450" color="white" frame="true" align="right" spinBox="true" caption="1alg" /> '''SOLUTION STRUCTURE OF AN HGR INHIBITOR, NMR...)
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Revision as of 13:53, 12 November 2007
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SOLUTION STRUCTURE OF AN HGR INHIBITOR, NMR, 10 STRUCTURES
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Overview
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.
Disease
Known disease associated with this structure: Hemolytic anemia due to glutathione reductase deficiency OMIM:[138300]
About this Structure
1ALG is a Single protein structure of sequence from Synthetic construct. Active as Glutathione-disulfide reductase, with EC number 1.8.1.7 Full crystallographic information is available from OCA.
Reference
Denaturation and reactivation of dimeric human glutathione reductase--an assay for folding inhibitors., Nordhoff A, Tziatzios C, van den Broek JA, Schott MK, Kalbitzer HR, Becker K, Schubert D, Schirmer RH, Eur J Biochem. 1997 Apr 15;245(2):273-82. PMID:9151953
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