The archaeal “7 kDa DNA-binding” proteins: extended characterization of an old gifted family

Abstract

The “7 kDa DNA-binding” family, also known as the Sul7d family, is composed of chromatin proteins from the Sulfolobales archaeal order. Among them, Sac7d and Sso7d have been the focus of several studies with some characterization of their properties. Here, we studied eleven other proteins alongside Sac7d and Sso7d under the same conditions. The dissociation constants of the purified proteins for binding to double-stranded DNA (dsDNA) were determined in phosphate-buffered saline at 25 °C and were in the range from 11 μM to 22 μM with a preference for G/C rich sequences. In accordance with the extremophilic origin of their hosts, the proteins were found highly stable from pH 0 to pH 12 and at temperatures from 85.5 °C to 100 °C. Thus, these results validate eight putative “7 kDa DNA-binding” family proteins and show that they behave similarly regarding both their function and their stability among various genera and species. As Sac7d and Sso7d have found numerous uses as molecular biology reagents and artificial affinity proteins, this study also sheds light on even more attractive proteins that will facilitate engineering of novel highly robust reagents.

A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM

“…All CD spectra were characteristic of mostly β‐stranded proteins with an α‐helix contribution as previously reported for Sul7d proteins (Edmondson & Shriver, ). The shape of these spectra correlates well with the one of Aho7c (Kalichuk et al, ), however a shift of the ellipticity minima towards a higher wavelength (228 nm) was observed for B10. In order to investigate whether mutants A2, B10, and C7 adopted the pH stability of Aho7c, these novel Affitins were incubated overnight at pH from 0 to 14.…”

“…We have previously described Affitins as artificial affinity proteins derived from the archaeal extremophilic 7 kDa DNA‐binding proteins, such as Sac7d and Sso7d (Béhar et al, ; Krehenbrink et al, ; Mouratou et al, ; Pecorari & Alzari, ). Recently, we have also extensively characterized this family of proteins (Kalichuk et al, ). Here we report the successful selection of binders derived from Aho7c, the smallest member of this family, identified to date.…”

“…The DNA library was created by saturation mutagenesis of the 10 codons coding for the amino acid residues in positions 9, 10, 22, 23, 25, 32, 34, 41, 43, and 45, which are expected to participate in the interaction between DNA and Aho7c (Kalichuk et al, ). The protocol has previously been described for Sac7d (Correa et al, ; Mouratou, Béhar, Paillard‐Laurance, Colinet, & Pecorari, ).…”

“…We have previously described such non‐antibody scaffolds: the Affitins (for artificial affinity proteins), which are based on members of the “7 kDa DNA‐binding” protein family, also known as Sul7d family (Krehenbrink et al, ; Mouratou et al, ; Pecorari & Alzari, ). These Sul7d proteins are found in various Archaea such as Sulfolobus , Acidianus , and Metallospharea genera, and are resistant to high temperatures (up to 90–100°C) and pH (0–12) (Kalichuk et al, ). Affitins, originally derived from Sac7d ( Sulfolobus acidocaldarius ) and Sso7d ( Sulfolobus solfataricus ), inherit the favorable properties of their parental proteins: they are stable (temperature and pH) and show comparable affinity/specificity as those of antibodies, being twenty times smaller (7 kDa compared to 150 kDa) at the same time.…”

“…It is of great interest to discover smaller scaffolds which are stable and soluble. As a step in that direction, we have recently characterized 13 members of the “7 kDa DNA‐binding” family, among which eight putative proteins (Kalichuk et al, ). One of these proteins, Aho7c, originating from Acidianus hospitalis , was shown to possess thermal stability comparable with Sso7d (96.8°C vs. 96.5°C) and higher than the one of Sac7d (89.6°C).…”