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Published on: September 26, 2015
Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen
Nikolai A Pavlov1, Dmitry I Cherny, Igor V Nazimov
1Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany.
Abstract:
Three novel DNA-binding proteins with apparent molecular masses of 7, 10 and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus kandleri. The proteins were identified using a blot overlay assay that was modified to emulate the high ionic strength intracellular environment of M.kandleri proteins. A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia coli. As indicated by CD spectroscopy and computer-assisted structure prediction methods, 7kMk is a substantially alpha-helical protein possibly containing a short N-terminal beta-strand. According to analytical gel filtration chromatography and chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further oligomerization. Electron microscopy showed that 7kMk bends DNA and also leads to the formation of loop-like structures of approximately 43.5 +/- 3.5 nm (136 +/- 11 bp for B-form DNA) circumference. A topoisomerase relaxation assay demonstrated that looped DNA is negatively supercoiled under physiologically relevant conditions (high salt and temperature). A BLAST search did not yield 7kMk homologs at the amino acid sequence level, but based on a multiple alignment with ribbon-helix-helix (RHH) transcriptional regulators, fold features and self-association properties of 7kMk we hypothesize that it could be related to RHH proteins.
Insights
Novel DNA-binding proteins were isolated from Methanopyrus kandleri. A 7 kDa protein, 7kMk, bends DNA into loops, which become negatively supercoiled, suggesting a role in DNA topology regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- The hyperthermophilic archaeon Methanopyrus kandleri possesses unique proteins adapted to extreme environments.
- Understanding DNA-binding proteins is crucial for deciphering DNA replication, repair, and regulation in extremophiles.
Purpose of the Study:
- To isolate and characterize novel DNA-binding proteins from M. kandleri.
- To investigate the structural and functional properties of a specific 7 kDa DNA-binding protein, designated 7kMk.
Main Methods:
- Blot overlay assay adapted for high ionic strength conditions.
- Cloning and expression of the 7kMk protein in Escherichia coli.
- Circular dichroism (CD) spectroscopy, computer-assisted structure prediction, analytical gel filtration, chemical crosslinking, electron microscopy, and topoisomerase relaxation assays.
Main Results:
- Three novel DNA-binding proteins were identified, including a 7 kDa protein (7kMk).
- 7kMk is an alpha-helical protein that forms stable dimers and higher-order oligomers.
- 7kMk induces DNA bending and the formation of DNA loops, leading to negative supercoiling under physiological conditions.
- No direct amino acid sequence homologs were found, but structural similarities suggest a potential relationship to ribbon-helix-helix (RHH) proteins.
Conclusions:
- The 7 kDa DNA-binding protein 7kMk from M. kandleri possesses unique DNA-bending and loop-forming capabilities.
- These activities result in DNA negative supercoiling, indicating a potential role in managing DNA topology in hyperthermophilic archaea.
- Despite lacking sequence homology, 7kMk's structural and functional characteristics suggest a possible evolutionary link to RHH transcriptional regulators.
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