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Biotinylation in the hyperthermophile Aquifex aeolicus
David J Clarke1, Joseph Coulson, Ranald Baillie
1School of Chemistry, University of Edinburgh, UK.
European Journal of Biochemistry
|March 13, 2003
Summary
Biotin protein ligase (BPL) from Aquifex aeolicus biotinylates biotin carboxyl carrier protein (BCCP) at high temperatures. Protein interactions reveal biotin addition reduces BCCP binding to BPL.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Biotin protein ligase (BPL) catalyzes specific biotinylation of biotin carboxyl carrier protein (BCCP).
- Understanding protein-protein interactions in hyperthermophiles like Aquifex aeolicus is crucial.
- Details on fatty acid roles in hyperthermophiles remain limited.
Purpose of the Study:
- To characterize the interaction between BPL and BCCP from Aquifex aeolicus.
- To investigate the role of biotinylation in BPL-BCCP complex formation.
- To elucidate the structural basis of substrate recognition in biotinylation.
Main Methods:
- Cloning, overexpression, and purification of recombinant BPL and BCCP Delta 67.
- In vitro biotinylation assays at high temperatures (up to 70°C).
- Limited proteolysis, chemical crosslinking (EDC), and mutant analysis (BCCP Delta 67 K117L).
Main Results:
- BPL successfully biotinylated BCCP Delta 67 at temperatures up to 70°C.
- Proteolysis of BPL was inhibited by MgATP and biotin, suggesting conformational changes.
- A BPL:apo-BCCP Delta 67 complex was isolated, with reduced formation for holo-BCCP Delta 67.
Conclusions:
- Biotinylation of BCCP Delta 67 by BPL involves specific protein-protein interactions.
- The addition of biotin to BCCP reduces its affinity for BPL.
- A network of interactions mediates recognition between BPL and BCCP, influenced by the biotinylation state.