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

Single-stranded DNA-binding proteins are recruited when single-stranded DNA is exposed by disruption of the duplex. Many important biological processe s 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 s o-called OB fold. The single-stranded DNA-binding protein of the crenarchae ote Sulfolobus solfataricus has a number of interesting differences and sim ilarities to both the eubacterial and eukaryotic homologues. It has an exte nded C-terminal tail with significant sequence identity to a similar region in the eubacterial protein. However, the sequence of the OB fold is much m ore like the eukaryotic and euryarchaeal proteins. The S. solfataricus prot ein remains a monomer in the absence of DNA but rapidly polymerizes upon bi nding - a behaviour not seen in the Escherichia coli protein. The protein h as been overexpressed, purified and crystallized. The protein crystallizes in two related forms, both having space group P6(1) (or P6(5)) with approxi mate unit-cell parameters a = b = 75, c = 69 Angstrom, but the crystals are distinguished by their size and morphology. The larger crystals are hexago nal bipyramids and are merohedrally twinned, diffracting to 1.34 Angstrom w ith diffraction observed to 1.2 Angstrom. Smaller needle-like crystals diff ract to about 2.0 Angstrom but are not twinned. Molecular-replacement attem pts have failed owing to low identity with available search models. The str ucture will be determined by multiple-wavelength methods.