Thermal Stability and DNA Binding Activity of a Variant Form of the Sso7d Protein from the Archeon Sulfolobus solfataricus Truncated at Leucine 54

Abstract

Sso7d is a 62-residue, basic protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Around neutral pH, it exhibits a denaturation temperature close to 100 degrees C and a non-sequence-specific DNA binding activity. Here, we report the characterization by circular dichroism and fluorescence measurements of a variant form of Sso7d truncated at leucine 54 (L54Delta). It is shown that L54Delta has a folded conformation at neutral pH and that its thermal unfolding is a reversible process, represented well by the two-state N <=> D transition model, with a denaturation temperature of 53 degrees C. Fluorescence titration experiments indicate that L54Delta binds tightly to calf thymus DNA, even though the binding parameters are smaller than those of the wild-type protein. Therefore, the truncation of eight residues at the C-terminus of Sso7d markedly affects the thermal stability of the protein, which nevertheless retains a folded structure and DNA binding activity.

Guanidine-induced unfolding of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus

“…The quality of the fit was good, as indicated by the small values of the standard deviation of the calculated points from the experimental ones. parameters obtained for Sso7d in the absence of GuHCl are close to those determined by means of differential scanning calorimetry, DSC, measurements by Ladenstein and co-workers [16], and those determined by CD measurements at 200 nm by us [29]. This is a strong indication that the temperature-induced unfolding of Sso7d is a two state N ⇔ D transition.…”

Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold

“…In the case of Sso7d, the hydrophobic core comprised of 11 residues (V3, F5, V14, I19, I29, F31, Y33, G43, V45, A50 and L54) as well as the C-terminal α helix play a major role in protein stability. 31,43 Mutagenesis of residues in the hydrophobic core results in a significant decrease in the melting temperature of Sso7d. 44 In particular, the single point mutation F31A was shown to decrease the melting temperature by 24°C.…”

“…45 Similarly, deletion of the C-terminal α helix causes a 46°C decrease in melting temperature. 43 The Sso7d mutants analyzed were also resistant to chemical denaturation mediated by Gdn-HCl and extreme conditions of pH. Of the mutants analyzed, Sso7d-β-catenin peptide has the lowest [Gdn-HCl] 1/2 of 2.5 M. By comparison, the corresponding [Gdn-HCl] 1/2 for a typical single chain antibody was reported as 1.5 M. 32 All Sso7d mutants analyzed could also withstand extended incubation in a wide range of pH values (0.33-12.5), while retaining their secondary structure; the data obtained at pH 13.5 were inconclusive.…”

Switching an anti-IgG binding site between archaeal extremophilic proteins results in Affitins with enhanced pH stability

“…Although initially mistakenly identified as OB-fold proteins Robinson et al, 1998), they both fold as an SH3-like five-stranded incomplete ␤-barrel capped by a C-terminal ␣-helix (Agback et al, 1998;Kahsai et al, 2005), and have closely related sequences (∼79% identity). Interestingly, Sso7d is about 10 • C more stable than Sac7d with a T m = 100.2 • C. While it is stable under acidic conditions as low as pH 0, the upper limit of its stability in extreme alkaline conditions is not known (Catanzano et al, 1998;Clark et al, 2004;Edmondson and Shriver, 2001;Shehi et al, 2003).…”