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Characterization of in vitro oxidized barstar
C Frisch1, G Schreiber, A R Fersht
1Cambridge Centre for Protein Engineering, Medical Research Council Centre, UK.
FEBS Letters
|August 21, 1995
Summary
Oxidizing barstar protein to form a disulfide bridge did not increase stability and abolished its inhibitory function against barnase. A mutation (E80A) partially restored stability in the oxidized form.
Area of Science:
- Biochemistry
- Protein Chemistry
- Molecular Biology
Background:
- Barstar is a polypeptide inhibitor of the ribonuclease barnase.
- Barstar possesses two cysteine residues (C40 and C82) potentially forming a stabilizing disulfide bridge.
- Previous hypotheses suggested this disulfide bridge enhances stability without altering inhibitory activity.
Purpose of the Study:
- To investigate the biochemical and physico-chemical properties of oxidized barstar and its E80A mutant.
- To determine the effect of disulfide bond formation on barstar's stability and inhibitory function.
- To analyze the structural integrity of oxidized barstar, particularly in relation to the E80A mutation.
Main Methods:
- In vitro oxidation of wild-type barstar and E80A mutant.
- Biochemical assays, including plate assays for barnase inhibition.
- Physico-chemical analyses, including Circular Dichroism (CD) spectroscopy.
Main Results:
- Oxidized barstar monomers exhibited no inhibition of barnase activity.
- Oxidized barstar showed significant destabilization and loss of secondary structure, confirmed by CD spectra.
- The E80A substitution stabilized oxidized barstar, comparable to its effect on the reduced form, suggesting local helical integrity.
Conclusions:
- The proposed disulfide bridge in barstar does not enhance stability and impairs inhibitory function.
- Oxidation leads to protein destabilization and loss of secondary structure.
- The E80A mutation offers partial stabilization to oxidized barstar, indicating the importance of residue 80 in maintaining structural integrity.