@article{487927f5-a2bc-4ce1-a9e2-487178c495fd,
  abstract     = {{<p>We present the structural and functional characterization of a single-stranded DNA-binding protein (SSB-M5) identified from a hot spring metagenome in Vatnajökull National Park, Iceland. This small protein (136 aa; 15,695 Da) shares 100% amino acid sequence identity with two previously uncharacterized SSBs from hyperthermophilic Fervidobacterium species. Functional complementation assay demonstrated that SSB-M5 can substitute for Escherichia coli SSB in an ssb<sup>−</sup> mutant strain, confirming its biological activity. A recombinant C-terminally His-tagged SSB-M5 was overproduced, purified to homogeneity, and subjected to structural, biochemical, and biophysical analysis. The crystal structure revealed that SSB-M5 forms a dimer through a crystallographic twofold axis, with each monomer contributing to a large antiparallel β-sheet. The flat surfaces of the β-sheets from the two dimers are packed together via a second crystallographic twofold axis, forming a tetramer that serves as the functional unit of the SSB-M5. Electrophoretic mobility shift assays showed that SSB-M5, after heat treatment up to 100°C, forms stable DNA-protein complexes with the (dT)<sub>40</sub> oligo. Quantitative analyses revealed that SSB-M5 binds (dT)<sub>70</sub> oligonucleotide with very high affinity (K<sub>D</sub> = 72 ± 6 pM). Hill analysis indicated cooperative binding, yielding an EC<sub>50</sub> of 141 pM and a Hill coefficient of 2. Moreover, inclusion of SSB-M5 in PCR reactions significantly enhanced amplification by eliminating non-specific products. Together, these findings identify SSB-M5 as a hyperthermostable, high-affinity single-stranded DNA-binding protein with potential applications in molecular biology and biotechnology.</p>}},
  author       = {{Werbowy, Olesia and Håkansson, Maria and Dorawa, Sebastian and Stefańska-Kaźmierczak, Aleksandra and Svensson, L. Anders and Al-Karadaghi, Salam and Jurczak-Kurek, Agata and Kwiatkowska-Semrau, Karolina and Plotka, Magdalena and Fridjonsson, Olafur H. and Hreggvidsson, Gudmundur O. and Aevarsson, Arnthór and Dąbrowski, Sławomir and Kaczorowska, Anna Karina and Kaczorowski, Tadeusz}},
  issn         = {{0961-8368}},
  keywords     = {{crystal structure; genetic complementation; PCR; SSB; thermal stability}},
  language     = {{eng}},
  number       = {{4}},
  publisher    = {{The Protein Society}},
  series       = {{Protein Science}},
  title        = {{Structural and functional characterization of a hyperthermostable single-stranded DNA-binding protein from a hot spring metagenome}},
  url          = {{http://dx.doi.org/10.1002/pro.70538}},
  doi          = {{10.1002/pro.70538}},
  volume       = {{35}},
  year         = {{2026}},
}