Sedimentation analysis of <i>Neq</i>SSB-like (A) and standard proteins (B).

<p>The proteins were analyzed on a 12% polyacrylamide gel. Lane M: Unstained Protein Weight Marker (Fermentas, Lithuania), with the molecular mass of proteins marked. Lane 1–19: fraction number. 50 μl of 150 μM <i>Neq</i>SSB-like and the corresponding amounts of standard proteins were centrifuged in linear 15 to 30% (w/v) glycerol gradients, as described in the “Methods”. The fractions with proteins were analyzed by SDS-PAGE. The fractions at which the maximal amount of protein appears are shown by arrows in each panel. The standard proteins used are: L, lysozyme (14 kDa); CA, carbonic anhydrase (29 kDa); BSA, bovine serum albumin (66 kDa) and AD, alcohol dehydrogenase (150 kDa). The oligomerization state estimation of <i>Neq</i>SSB-like was made with these proteins.</p

Binding of <i>Neq</i>SSB-like to M13 ssDNA.

<p>Lanes 1–8 contain (0.07 pmol) of M13 ssDNA and 0, 3.5, 7, 14, 28, 56, 112 and 224 pmoles of <i>Neq</i>SSB-like, respectively.</p

<i>Neq</i>SSB-like dsDNA and mRNA binding properties.

<p><b>A</b> Binding to 2.5 pmol of 100 bp PCR product. Lanes 1–6 contain 0, 10, 20, 40, 80 and 160 pmoles of <i>Neq</i>SSB-like, respectively. <b>B</b> Binding to 0.132 pmol of <i>Escherichia coli</i> genomic DNA. Lanes 1–7 contain 0, 10, 20, 40, 80, 160 and 320 pmoles of <i>Neq</i>SSB-like, respectively. <b>C</b> Binding to 0.2 pmol of pDONR201 plasmid DNA (4470 bp). Lanes 1–7 contain 0, 10, 20, 40, 80, 160 and 320 pmoles of <i>Neq</i>SSB-like, respectively. <b>D</b> Binding to 0.1 pmol of pDONR201 plasmid DNA + 0.05 pmol of linearized pDONR201 plasmid DNA. Lanes 1–7 contain 0, 10, 20, 40, 80, 160 and 320 pmoles of <i>Neq</i>SSB-like, respectively. <b>E</b> Control binding reaction with 0.2 pmol of pDONR201 plasmid DNA. Lanes 1–4 contain 0, 10, 20 and 40 pmoles of <i>Taq</i>SSB, respectively. <b>F</b> Binding to 980 ng of mRNA. Lanes 1–5 contain 0, 10, 20, 40, 80 pmoles of <i>Neq</i>SSB-like, respectively.</p

<i>Neq</i>SSB-like binding preferences.

<p>The reactions contained a fixed quantity of the sample DNA: 10 pmol of (dT)₇₆ and 2.5 pmol of 100 bp PCR product. Lane 1: (dT)₇₆ with 0 pmol of <i>Neq</i>SSB-like. Lane 2: 100 bp with 0 pmol of <i>Neq</i>SSB-like. Lane 3: (dT)₇₆ and 100 bp PCR product with 0 pmol of <i>Neq</i>SSB-like. Lanes 4–9 contain 10, 20, 40, 80, 160 and 320 pmoles of <i>Neq</i>SSB-like, respectively.</p

Interaction analysis of <i>Neq</i>SSB-like (A) with ssDNA and dsDNA (B).

<p>Different concentrations of the protein were injected with a flow rate of 30 μl/min on a streptavidin chip coated with ssDNA 60-mer and dsDNA 60-mer on separate flow channels. A flow cell with streptavidin was used as a reference. After each injection the chip was regenerated with 0.01% SDS. The different colors of the sensograms represent the concentrations of the <i>Neq</i>SSB-like injected. Solid lines state for fitted curves. The data were fitted in accordance with the Langmuir model and using BiaEval 3.0 software.</p

The determination of ssDNA-binding activity half-life, using gel mobility shift assays, for <i>Neq</i>SSB-like and <i>Taq</i>SSB.

<p><i>Neq</i>SSB-like represented by the open circles and <i>Taq</i>SSB, shown as open triangles.</p

Results of the analytical gel filtration of <i>Neq</i>SSB-like on the Superdex 75 10/300 GL column.

<p><b>A</b> The standard linear regression curve was generated by plotting the log of the molecular mass of calibration proteins against V_e/V₀ value, namely elution volume divided by void volume. The calibration proteins represented by black triangles include bovine albumin (66 kDa), ovalbumin (43 kDa), carbon anhydrase (29 kDa) and ribonuclease A (13 kDa). <i>Neq</i>SSB-like is represented by the black circle. The regression curve equation and coefficient of determination are shown. <b>B</b> Effects of <i>Neq</i>SSB-like protein concentrations on the elution profiles of gel filtration. The entire range of <i>Neq</i>SSB-like concentrations (12–236 μM) show an elution volume of 15.8 ml corresponding to the monomeric protein.</p

The multiple amino acid alignment.

<p><b>A</b> The multiple amino acid alignment of nucleic acid binding protein from <i>Nanoarchaeum equitans</i>, bacterial and craenarchaeal SSB proteins. <b>B</b> The multiple amino acid alignment of nucleic acid binding protein from <i>Nanoarchaeum equitans</i> and craenarchaeal SSB proteins. The alignments were performed by dividing the amino acids into six similarity groups: group 1 V, L, I, M, group 2 W, F, Y, group 3 E, D, group 4 K, R, group 5 Q, D, and group 6 S, T. The capital letters represent single amino acid codes. White fonts on black boxes represent 100% similarity, white fonts on grey boxes denote <80% similarity, and black fonts on grey boxes show <60% similarity. Abbreviations: NeqSSB—nucleic acid binding protein from <i>Nanoarchaeum equitans</i> Kin-4M, SsoSSB <i>Sulfolobus solfataricus</i> strain P2, SacSSB <i>Sulfolobus acidocaldarius</i> DSM 639, SmaSSB <i>Staphylothermus marinus</i> F1, DkeSSB <i>Desulfurococcus kamchatkensis</i> 1221n, EcoSSB <i>Escherichia coli</i> K12, TmaSSB <i>Thermotoga maritima</i> strain MSB8, and TteSSB3 <i>Thermoanerobacter tengcongensis</i> MB4. The W108 residue, important in base-stacking interactions is indicated in the panel B. The blue box indicates OB-fold region.</p

The expression and purification of <i>Neq</i>SSB-like from <i>E</i>. <i>coli</i> TOP10F’+pBAD/NeqSSBHT.

<p>The proteins were analyzed on a 12% polyacrylamide gel. Lane M: Unstained Protein Weight Marker (Fermentas, Lithuania), with the molecular mass of proteins marked. Lane 1: soluble protein cell extracts after arabinose induction of protein expression (10 μl). Lane 2: <i>Neq</i>SSB-like after the Ni²⁺-affinity chromatography step (10 μl). Lane 3: <i>Neq</i>SSB-like after His-tag cleavage with TEV protease (10 μl). Lane 4: <i>Neq</i>SSB-like after chromatography on an ssDNA-cellulose column (10 μl).</p

Binding of <i>Neq</i>SSB-like to a fixed quantity (10 pmol) of oligonucleotides.

<p><b>A</b> (dT)₃₅<b>B</b> (dT)₇₆<b>C</b> (dT)₁₂₀. Lanes 1–8 contain 0, 10, 20, 40, 80, 160, 320 and 640 pmoles of <i>Neq</i>SSB-like, respectively.</p