RESUMO
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.
Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Thermoplasma/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Sequência de Bases , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Clonagem Molecular , Primers do DNA , DNA Arqueal , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The algorithm [Bethanis, Tzamalis, Hountas & Tsoucaris (2002). Acta Cryst. A58, 265-269] which reformulates the quantum-mechanical problem of solving a Schrödinger (S) equation in a crystallographic context has been upgraded and tested for many aspects of convergence. The upgraded algorithm in reciprocal space aims at determining a wavefunction Phi(H) such that (a) Phi(H) fulfils the S equation within certain precision and (b) Phi(H) minimizes by least squares the differences between the calculated structure factors from the wavefunction and the observed ones. Calculations have been made with three molecules (11, 41 and 110 non-H atoms in the asymmetric unit) for different numbers of initially given phases. Three main questions have been addressed: (I) Does the iterative calculation of the wavefunction converge? (II) Do the calculated wavefunctions converge to a unique set of Phi(H) values independent of the initial random set of Phi(H)? (III) Is the calculated Phi(H) set a good approximation of a wavefunction able to produce within certain errors the correct values of the phases of the structure factors? Concerning questions (I) and (II), our results give a strong hint about fast convergence to a unique wavefunction independent of the arbitrary starting wavefunction. This is an essential prerequisite for practical applications. For question (III) in the case closer to the ab initio situation, the final mean phase error, respectively, for the three structures is 3, 26 and 28 degrees. The combination of (a) and (b) in the upgraded algorithm has been proved crucial especially for the results concerning the larger structures.