Determining the role of metal binding in protein cage assembly.

Assembly of protein cages may require structural metal ions to nucleate or stabilize association of protein subunits. We describe here an approach to establishing the role of metal ions in protein cage assembly and stability, focusing on detecting the presence of structural metal ions as well as establishing oligomeric state. A colorimetric assay for detection of metal is described, along with a gel filtration assay to assess protein cage assembly and a fluorescence-based method for determining protein stability.

Functional comparison of Deinococcus radiodurans Dps proteins suggests distinct in vivo roles.

Deinococcus radiodurans exhibits extreme resistance to DNA damage and is one of only few bacteria that encode two Dps (DNA protection during starvation) proteins. Dps-1 was shown previously to bind DNA with high affinity and to localize to the D. radiodurans nucleoid.

Metal binding at the Deinococcus radiodurans Dps-1 N-terminal metal site controls dodecameric assembly and DNA binding.

The prokaryotic DNA protection during starvation (Dps) proteins typically protect macromolecules against damaging agents via physical association with DNA and by oxidizing and sequestering iron. However, Deinococcus radiodurans Dps-1, which binds DNA with high affinity, fails to protect DNA against hydroxyl radicals due to iron leakage from the core, raising the question of how (•)OH-mediated damage to Dps-1-bound DNA is avoided. As shown here, Mn(II) inhibits ferroxidase activity, suggesting that ferroxidation may be prevented in vivo as D.

On the stoichiometry of Deinococcus radiodurans Dps-1 binding to duplex DNA.

DNA protection during starvation (Dps) proteins, dodecameric assemblies of four-helix bundle subunits, contribute to protection against reactive oxygen species. Deinococcus radiodurans, which is characterized by resistance to DNA damaging agents, encodes two Dps homologs, of which Dps-1 binds DNA with high affinity. DNA binding requires N-terminal extensions preceding the four-helix bundle core.