Characterization of in vitro oxidized barstar.
The polypeptide inhibitor of the ribonuclease barnase, barstar, has two cysteine residues in positions 40 and 82. These have been proposed to form a disulfide bridge leading to an increase in stability without changing the inhibitory activity of the protein. Barstar and a mutant (E80A) were oxidized in vitro and the biochemical and physico-chemical properties of the oxidized monomers were analysed. The oxidized proteins show no inhibition of barnase using a plate assay and are significantly destabilized. CD spectra indicate a loss of secondary structure. The amino acid substitution E80 --> A stabilizes the oxidized barstar to about the same extent as it does the reduced protein, indicating, however, that the helical region which it is in is intact.
Frisch
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Cambridge Centre for Protein Engineering, Medical Research Council Centre, UK.
Schreiber
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D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D013696:Temperature; D014508:Urea
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; C057438:barstar protein, Bacillus amyloliquefaciens; D014508:Urea; D012260:Ribonucleases