The stability of the archaeal HU histone-like DNA-binding protein from Thermoplasma volcanium.
The complete genome analysis of the archaeon Thermoplasma volcanium has revealed a gene assigned to encode the histone-like DNA-binding protein HU. Thermoplasma volcanium is a moderate thermophile growing around 60 degrees C and it is adaptable to aerobic and anaerobic environment and therefore it is unique as a candidate for the origin of eukaryotic nuclei in the endosymbiosis hypothesis. The HU protein is the major component of the bacterial nuclei and therefore it is an important protein to be studied. The gene for HUTvo protein (huptvo) was cloned from the genomic DNA of T. volcanium and overexpressed in Escherichia coli. A fast and efficient purification scheme was established to produce an adequate amount of bioactive protein for biochemical and biophysical studies. Highly purified HUTvo was studied for its DNA-binding activity and thermostability. As studied by circular dichroism and high-precision differential scanning microcalorimetry, the thermal unfolding of HUTvo protein is reversible and can be well described by a two-state model with dissociation of the native dimeric state into denatured monomers. The G versus T profile for HUTvo compared to the hyperthermophilic marine eubacterial counterpart from Thermotoga maritima, HUTmar, clearly shows that the archaeal protein has adopted a less efficient molecular mechanism to cope with high temperature. The molecular basis of this phenomenon is discussed.
Orfaniotou
Fotini
F
Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis-Zographou, 15784 Athens, Greece.
Tzamalis
Pavlos
P
Thanassoulas
Angelos
A
Stefanidi
Eleni
E
Zees
Athanassios
A
Boutou
Effrosini
E
Vlassi
Metaxia
M
Nounesis
George
G
Vorgias
Constantinos E
CE
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D019641:DNA, Archaeal; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013822:Thermoplasma / Q000378:metabolism*
D019843:Archaeal Proteins; D017931:DNA Primers; D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins