Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans--A Nucleic Acid Binding Protein with Broad Substrate Specificity

Abstract

Background: SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.

Results: This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7 ± 1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100 °C and melting temperature (T(m)) is 100.2 °C as shown by differential scanning calorimetry (DSC) analysis.

Conclusion: NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.

Fig 1. The multiple amino acid alignment.

A The multiple amino acid alignment of nucleic acid binding protein from Nanoarchaeum equitans, bacterial and craenarchaeal SSB proteins. B The multiple amino acid alignment of nucleic acid binding protein from Nanoarchaeum equitans 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 Nanoarchaeum equitans Kin-4M, SsoSSB Sulfolobus solfataricus strain P2, SacSSB Sulfolobus acidocaldarius DSM 639, SmaSSB Staphylothermus marinus F1, DkeSSB Desulfurococcus kamchatkensis 1221n, EcoSSB Escherichia coli K12, TmaSSB Thermotoga maritima strain MSB8, and TteSSB3 Thermoanerobacter tengcongensis MB4. The W108 residue, important in base-stacking interactions is indicated in the panel B. The blue box indicates OB-fold region.

Fig 2. The expression and purification of NeqSSB-like from E. coli TOP10F’+pBAD/NeqSSBHT.

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: NeqSSB-like after the Ni²⁺-affinity chromatography step (10 μl). Lane 3: NeqSSB-like after His-tag cleavage with TEV protease (10 μl). Lane 4: NeqSSB-like after chromatography on an ssDNA-cellulose column (10 μl).

Fig 3. Results of the analytical gel filtration of NeqSSB-like on the Superdex 75 10/300 GL column.

A 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). NeqSSB-like is represented by the black circle. The regression curve equation and coefficient of determination are shown. B Effects of NeqSSB-like protein concentrations on the elution profiles of gel filtration. The entire range of NeqSSB-like concentrations (12–236 μM) show an elution volume of 15.8 ml corresponding to the monomeric protein.

Fig 4. Sedimentation analysis of NeqSSB-like (A) and standard proteins (B).

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 NeqSSB-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 NeqSSB-like was made with these proteins.

Fig 5. Binding of NeqSSB-like to a fixed quantity (10 pmol) of oligonucleotides.

A (dT)₃₅ B (dT)₇₆ C (dT)₁₂₀. Lanes 1–8 contain 0, 10, 20, 40, 80, 160, 320 and 640 pmoles of NeqSSB-like, respectively.

Fig 6. Binding of NeqSSB-like to M13 ssDNA.

Lanes 1–8 contain (0.07 pmol) of M13 ssDNA and 0, 3.5, 7, 14, 28, 56, 112 and 224 pmoles of NeqSSB-like, respectively.

Fig 7. The inverse fluorescence titration of NeqSSB-like with poly(dT).

2 nM samples were titrated with a saturating quantity of poly(dT) in 2 mM, represented by the fill diamonds, 100 mM, shown as fill triangles and 500 mM NaCl, given as open squares, in a binding buffer. The vertical lines indicate the binding site size calculated for each assay.

Fig 8. NeqSSB-like dsDNA and mRNA binding properties.

A Binding to 2.5 pmol of 100 bp PCR product. Lanes 1–6 contain 0, 10, 20, 40, 80 and 160 pmoles of NeqSSB-like, respectively. B Binding to 0.132 pmol of Escherichia coli genomic DNA. Lanes 1–7 contain 0, 10, 20, 40, 80, 160 and 320 pmoles of NeqSSB-like, respectively. C 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 NeqSSB-like, respectively. D 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 NeqSSB-like, respectively. E Control binding reaction with 0.2 pmol of pDONR201 plasmid DNA. Lanes 1–4 contain 0, 10, 20 and 40 pmoles of TaqSSB, respectively. F Binding to 980 ng of mRNA. Lanes 1–5 contain 0, 10, 20, 40, 80 pmoles of NeqSSB-like, respectively.

Fig 9. NeqSSB-like binding preferences.

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 NeqSSB-like. Lane 2: 100 bp with 0 pmol of NeqSSB-like. Lane 3: (dT)₇₆ and 100 bp PCR product with 0 pmol of NeqSSB-like. Lanes 4–9 contain 10, 20, 40, 80, 160 and 320 pmoles of NeqSSB-like, respectively.

Fig 10. Interaction analysis of NeqSSB-like (A) with ssDNA and dsDNA (B).

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 NeqSSB-like injected. Solid lines state for fitted curves. The data were fitted in accordance with the Langmuir model and using BiaEval 3.0 software.

Fig 11. The determination of ssDNA-binding activity half-life, using gel mobility shift assays, for NeqSSB-like and TaqSSB.

NeqSSB-like represented by the open circles and TaqSSB, shown as open triangles.

Fig 12. NeqSSB-like thermogram.

A sample containing 1 mg/ml of purified protein was analyzed in a 20 mM phosphate buffer pH 7.5 containing 150 mM NaCl. NeqSSB-like melting temperature is shown.