DNA-binding specificity of the Lon protease α-domain from Brevibacillus thermoruber WR-249
Date Issued
2010
Date
2010
Author(s)
Lin, Yu-Ching
Abstract
The multi-functional, homo-oligomeric, ATP-dependent Lon protease is highly conserved in prokaryotes and eukaryotic organelles. Previous studies have shown that Lon activity is essential for protein quality control and regulation of metabolic processes. Here we examined the DNA-binding activity of the Lon protease α-domains from Brevibacillus thermoruber, Bacillus subtilis, and Escherichia coli. Gel mobility shift assays indicated that the α-domain from Br. thermoruber has the highest DNA affinity. MALDI-TOF mass spectrometry showed that this α-domain binds to the nucleotide sequence 5’-CTGTTAGCGGGC-3’ (ms1). Surface plasmon resonance and isothermal titration calorimetry showed that a double-stranded DNA fragment of this sequence binds to the α-domain; double-stranded DNA fragments with 0 and 50% identity to the binding sequence had lower affinities for the α-domain. Five mutants of the α-domain from Br. thermoruber carrying single mutations (R537A, R546A, R553A, K580A and R584A) were constructed and showed only 1.2–2.0-fold lower DNA binding affinity; one mutant, R518A, displayed 26-fold lower affinity. The Bt-Lon R518A mutant also has lower affinity to DNA than wild type. These results revealed that Arg 518 of the Bt-Lon from Br. thermoruber plays a critical role in the DNA-binding activity.
Subjects
Brevibacillus thermoruber WR-249
Lon
α-domain
Surface Plasmon resonance
Isothermal titration calorimetry
Type
thesis
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