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Fluorescence thiol modification assay: oxidatively modified proteins in Bacillus subtilis
Falko Hochgräfe1, Jörg Mostertz, Dirk Albrecht
1Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität Greifswald, F.-L.-Jahn-Strasse 15, D-17487 Greifswald, Germany.
Molecular Microbiology
|October 1, 2005
Summary
Bacterial protein thiols are mostly reduced, but oxidative stress from agents like hydrogen peroxide or antibiotics oxidizes specific enzymes. This thiol oxidation can lead to methionine limitation by inactivating key enzymes.
Area of Science:
- Biochemistry
- Microbiology
- Proteomics
Background:
- Oxidative modification of thiol groups in cysteine residues impacts protein activity.
- Understanding the in vivo thiol state of proteins is crucial for cellular function.
Purpose of the Study:
- To develop and apply a method for monitoring the in vivo thiol state of cytoplasmic proteins.
- To identify proteins sensitive to oxidative stress in Bacillus subtilis.
Main Methods:
- Development of a fluorescence-based thiol modification assay.
- Combination with two-dimensional gel electrophoresis and mass spectrometry.
- Exposure of Bacillus subtilis to oxidative stressors (diamide, hydrogen peroxide, paraquat) and antibiotic (nitrofurantoin).
Main Results:
- In growing Bacillus subtilis, protein thiols are predominantly in a reduced state.
- Diamide treatment significantly increased oxidized thiols in metabolic enzymes.
- Hydrogen peroxide primarily oxidized proteins with active site cysteines, affecting enzymes like methionine synthase (MetE), leading to methionine limitation.
Conclusions:
- The study provides insights into the dynamic thiol redox state of proteins in Bacillus subtilis under various stress conditions.
- Oxidative stress can lead to inactivation of essential enzymes, impacting cellular metabolism.
- The antibiotic nitrofurantoin was shown to induce thiol oxidation in B. subtilis.