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Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy
Published on: August 20, 2018
Regulation of FNR dimerization by subunit charge repulsion
Laura J Moore1, Erin L Mettert, Patricia J Kiley
1Department of Chemistry, Monmouth College, Monmouth, Illinois 61462, USA. lmoore@monm.edu
The Journal of Biological Chemistry
|September 9, 2006
Summary
Charge repulsion from specific amino acids inhibits the dimerization of the anaerobic transcription factor FNR under aerobic conditions. A [4Fe-4S] cluster is needed for dimerization under anaerobic conditions.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Biochemistry
Background:
- Dimerization of the FNR transcription factor is crucial for its activity.
- Under aerobic conditions, FNR is monomeric and inactive due to the absence of its oxygen-labile [4Fe-4S] cluster.
Purpose of the Study:
- To identify protein side chains that prevent FNR dimerization under aerobic conditions.
- To understand the mechanism of FNR regulation by oxygen and its [4Fe-4S] cluster.
Main Methods:
- Site-directed mutagenesis of FNR, specifically at positions 150 and 154.
- In vitro analysis of FNR mutant protein activity.
- Protease sensitivity assays (trypsin).
- Circular dichroism spectroscopy.
Main Results:
- Substituting Asp(154) or Glu(150) with neutral or positively charged residues increased FNR activity under aerobic conditions via an [4Fe-4S]-independent mechanism.
- Simultaneous substitution of residues 150 and 154 with Lys restored aerobic inhibition.
- Small changes in secondary structure were observed between cluster-containing FNR and apoFNR, but protease sensitivity remained similar.
Conclusions:
- Charge repulsion between amino acid residues at positions 150 and 154 is essential for inhibiting FNR dimerization under aerobic conditions.
- A [4Fe-4S]-dependent conformational change, involving subtle structural alterations, likely overcomes this charge repulsion to enable dimerization under anaerobic conditions.
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