Structural and functional characterization of a hyperthermostable single‐stranded DNA ‐binding protein from a hot spring metagenome
Year: 2026
Type: article
Abstract: 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 − 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) 40 oligo. Quantitative analyses revealed that SSB‐M5 binds (dT) 70 oligonucleotide with very high affinity ( K D = 72 ± 6 pM). Hill analysis indicated cooperative binding, yielding an EC 50 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.
Source: Protein Science
DOI: 10.1002/pro.70538
Authors: Olesia Werbowy, Maria Håkansson, Sebastian Dorawa, Aleksandra Stefańska‐Kaźmierczak, L. Anders Svensson, Salam Al‐Karadaghi, Agata Jurczak‐Kurek, Karolina Kwiatkowska‐Semrau, Magdalena Plotka, Olafur H. Fridjonsson, Gudmundur O. Hreggvidsson, Arnthór Aevarsson, Sławomir Dąbrowski, Anna‐Karina Kaczorowska, Tadeusz Kaczorowski
Language: English
Subfield: Materials Chemistry
Field: Materials Science
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7140116818