Structural and functional characterization of a hyperthermostable single-stranded DNA-binding protein from a hot spring metagenome
(2026) In Protein Science 35(4).- 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... (More)
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 (KD = 72 ± 6 pM). Hill analysis indicated cooperative binding, yielding an EC50 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.
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- author
- publishing date
- 2026-04
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- crystal structure, genetic complementation, PCR, SSB, thermal stability
- in
- Protein Science
- volume
- 35
- issue
- 4
- article number
- e70538
- publisher
- The Protein Society
- external identifiers
-
- scopus:105034115097
- pmid:41870280
- ISSN
- 0961-8368
- DOI
- 10.1002/pro.70538
- language
- English
- LU publication?
- no
- additional info
- Publisher Copyright: © 2026 The Protein Society.
- id
- 487927f5-a2bc-4ce1-a9e2-487178c495fd
- date added to LUP
- 2026-05-25 16:14:06
- date last changed
- 2026-09-16 07:03:40
@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}},
}