Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri.
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.
Pavlov
Nikolai A
NA
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany.
Cherny
Dmitry I
DI
Nazimov
Igor V
IV
Slesarev
Alexei I
AI
Subramaniam
Vinod
V
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D019641:DNA, Archaeal / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D019605:Euryarchaeota / Q000737:chemistry / Q000235:genetics*; D008854:Microscopy, Electron; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D016415:Sequence Alignment; D017421:Sequence Analysis; D012984:Software
D003432:Cross-Linking Reagents; D019641:DNA, Archaeal; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D009707:Nucleosomes; D011994:Recombinant Proteins; D004264:DNA Topoisomerases, Type I