Over-expression, purification, crystallization and data collection of a single stranded DNA binding protein from Sulfolobus solfataricus

ID Kerr, RIM Wadsworth, W Blankenfeldt, AG Staines, Malcolm Frederick White, James Henderson Naismith

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

Single-stranded DNA-binding proteins are recruited when single-stranded DNA is exposed by disruption of the duplex. Many important biological processes such as DNA replication can only occur when the two strands of the duplex are separated. A defining trait of these proteins is the presence of the so-called OB fold. The single-stranded DNA-binding protein of the crenarchaeote Sulfolobus solfataricus has a number of interesting differences and similarities to both the eubacterial and eukaryotic homologues. It has an extended C-terminal tail with significant sequence identity to a similar region in the eubacterial protein. However, the sequence of the OB fold is much more like the eukaryotic and euryarchaeal proteins. The S. solfataricus protein remains a monomer in the absence of DNA but rapidly polymerizes upon binding - a behaviour not seen in the Escherichia coli protein. The protein has been overexpressed, purified and crystallized. The protein crystallizes in two related forms, both having space group P6(1) (or P6(5)) with approximate unit-cell parameters a = b = 75, c = 69 Angstrom, but the crystals are distinguished by their size and morphology. The larger crystals are hexagonal bipyramids and are merohedrally twinned, diffracting to 1.34 Angstrom with diffraction observed to 1.2 Angstrom. Smaller needle-like crystals diffract to about 2.0 Angstrom but are not twinned. Molecular-replacement attempts have failed owing to low identity with available search models. The structure will be determined by multiple-wavelength methods.

Original languageEnglish
Pages (from-to)1290-1292
Number of pages3
JournalActa Crystallographica. Section D, Biological crystallography
VolumeD57
Publication statusPublished - Sept 2001

Keywords

  • ESCHERICHIA-COLI
  • CRYSTAL-STRUCTURE
  • SSB
  • COMPLEX
  • ARCHAEA
  • DOMAIN
  • SSDNA

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