Dps–DNA interaction in Marinobacter hydrocarbonoclasticus protein: effect of a single-charge alteration

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

Abstract

DNA-binding proteins from starved cells (Dps) are members of the ferritin family of proteins found in prokaryotes, with hollow rounded cube-like structures, composed of 12 equal subunits. These protein nanocages are bifunctional enzymes that protect the cell from the harmful reaction of iron and peroxide (Fenton reaction), thus preventing DNA damage by oxidative stress. Ferrous ions are oxidized at specific iron-binding sites in the presence of the oxidant and stored in its cavity that can accommodate up to ca. 500 iron atoms. DNA-binding properties of Dps are associated with the N-terminal, positive charge rich, extensions that can promote DNA binding and condensation, apparently by a cooperative binding mechanism. Here, we describe the binding and protection activities of Marinobacter hydrocarbonoclasticus Dps using Electrophoretic Mobility Shift Essays (EMSA), and synchrotron radiation circular dichroism (SRCD) spectroscopy. While no DNA condensation was observed in the tested conditions, it was possible to determine a Dps–DNA complex formation with an apparent dissociation constant of 6.0 ± 1.0 µM and a Hill coefficient of 1.2 ± 0.1. This interaction is suppressed by the inclusion of a single negative charge in the N-terminal region by point mutation. In Dps proteins containing a ferric mineral core (above 96 Fe/protein), DNA binding was impaired. SRCD data clearly showed that no significant modification existed either in secondary structure or protein stability of WT, Q14E variant and core containing proteins. It was, however, interesting to note that, in our experimental conditions, thermal denaturation induced protein aggregation that caused artifacts in thermal denaturation curves, which were dependent on radiation flux and vertical arrangement of the CD cell.

Original languageEnglish
Pages (from-to)513-521
Number of pages9
JournalEuropean Biophysics Journal
Volume50
Issue number3-4
DOIs
Publication statusPublished - May 2021

Keywords

  • DNA-binding protein from starved cells
  • Protein nanocages
  • Synchrotron radiation circular dichroism
  • Thermal stability

Fingerprint

Dive into the research topics of 'Dps–DNA interaction in Marinobacter hydrocarbonoclasticus protein: effect of a single-charge alteration'. Together they form a unique fingerprint.

Cite this