The HU protein from Thermotoga maritima: recombinant expression, purification and physicochemical characterization of an extremely hyperthermophilic DNA-binding protein
- PMID: 10518949
- DOI: 10.1006/jmbi.1999.3022
The HU protein from Thermotoga maritima: recombinant expression, purification and physicochemical characterization of an extremely hyperthermophilic DNA-binding protein
Abstract
The histone-like protein TmHU from the hyperthermophilic eubacterium Thermotoga maritima was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and cation exchange chromatography. CD spectroscopical studies with secondary structure analysis as well as comparative modeling demonstrate that the dimeric TmHU has a tertiary structure similar to other homologous HU proteins. The Tm of the protein was determined to be 96 degrees C, and thermal unfolding is nearly completely reversible. Surface plasmon resonance measurements for TmHU show that the protein binds to DNA in a highly cooperative manner, with a KD of 73 nM and a Hill coefficient of 7.6 for a 56 bp DNA fragment. It is demonstrated that TmHU is capable to increase the melting point of a synthetic, double-stranded DNA (poly[d(A-T)]) by 47 degrees C, thus suggesting that DNA stabilization may be a major function of this protein in hyperthermophiles. The significant in vitro protection of double-helical DNA may be useful for biotechnological applications.
Similar articles
-
High-affinity DNA binding of HU protein from the hyperthermophile Thermotoga maritima.J Mol Biol. 2001 Aug 17;311(3):491-502. doi: 10.1006/jmbi.2001.4763. J Mol Biol. 2001. PMID: 11493003
-
High-resolution X-ray structure of the DNA-binding protein HU from the hyper-thermophilic Thermotoga maritima and the determinants of its thermostability.Extremophiles. 2003 Apr;7(2):111-22. doi: 10.1007/s00792-002-0302-7. Epub 2002 Dec 12. Extremophiles. 2003. PMID: 12664263
-
Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant.Eur J Biochem. 2004 Apr;271(8):1497-507. doi: 10.1111/j.1432-1033.2004.04057.x. Eur J Biochem. 2004. PMID: 15066175
-
The physiological role of eubacterial histone-like proteins.Acta Microbiol Pol. 1989;38(3-4):207-15. Acta Microbiol Pol. 1989. PMID: 2484739 Review.
-
Recombinant DNA technology and crystallography. A new alliance in unraveling protein structure-function relationships.Bioprocess Technol. 1991;12:285-315. Bioprocess Technol. 1991. PMID: 1367088 Review. No abstract available.
Cited by
-
Functional roles of N-terminal and C-terminal domains in the overall activity of a novel single-stranded DNA binding protein of Deinococcus radiodurans.FEBS Open Bio. 2015 Apr 21;5:378-87. doi: 10.1016/j.fob.2015.04.009. eCollection 2015. FEBS Open Bio. 2015. PMID: 25973364 Free PMC article.
-
Optimization of expression and properties of the recombinant acetohydroxyacid synthase of Thermotoga maritima.Data Brief. 2015 Oct 3;5:489-97. doi: 10.1016/j.dib.2015.09.018. eCollection 2015 Dec. Data Brief. 2015. PMID: 26629492 Free PMC article.
-
An alternative flexible conformation of the E. coli HUβ₂ protein: structural, dynamics, and functional aspects.Eur Biophys J. 2011 Feb;40(2):117-29. doi: 10.1007/s00249-010-0630-y. Epub 2010 Oct 10. Eur Biophys J. 2011. PMID: 20936276
-
Bacterial nucleoid-associated proteins, nucleoid structure and gene expression.Nat Rev Microbiol. 2010 Mar;8(3):185-95. doi: 10.1038/nrmicro2261. Epub 2010 Feb 8. Nat Rev Microbiol. 2010. PMID: 20140026 Review.
-
DNA protection by histone-like protein HU from the hyperthermophilic eubacterium Thermotoga maritima.Nucleic Acids Res. 2008 Jul;36(12):3956-68. doi: 10.1093/nar/gkn348. Epub 2008 May 30. Nucleic Acids Res. 2008. PMID: 18515342 Free PMC article.
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
