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anderová1
lková1
1,2
í Jonák1
1 Department of Protein Biosynthesis, Institute of Molecular Genetics and
2 Department of Peptide Chemistry, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague 6, Czech Republic
Reprint requests to: Jií Jonák, Department of Protein Biosynthesis, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 37 Prague 6, Czech Republic; e-mail: jjon{at}img.cas.cz; fax: 420-224310955.
Recombinant mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) elongation factors EF-Tus, their isolated G-domains, and six chimeric EF-Tus composed of domains of either EF-Tu were prepared, and their GDP/GTP binding activities and thermostability were characterized. BstEF-Tu and BstG-domain bound GDP and GTP with affinities in nanomolar and submicromolar ranges, respectively, fully comparable with those of EcEF-Tu. In contrast, the EcG-domain bound the nucleotides with much lower, micromolar affinities. The exchange of domains 2 and 3 had essentially no effect on the GDP-binding activity; all complexes of chimeric EF-Tus with GDP retained Kd values in the nanomolar range. The final thermostability level of either EF-Tu was the result of a cooperative interaction between the G-domains and domains 2 + 3. The G-domains set up a "basic" level of the thermostability, which was ~20°C higher with the BstG-domain than with the EcG-domain. This correlated with the growth temperature optimum difference of both bacteria and two distinct thermostabilization features of the BstG-domain: an increase of charged residues at the expense of polar uncharged residues (CvP bias), and a decrease in the nonpolar solvent-accessible surface area. Domains 2 + 3 contributed by further stabilization of 
-helical regions and, in turn, the functions of the G-domains to the level of the respective growth temperature optima. Their contributions were similar irrespective of their origin but, with Ecdomains 2 + 3, dependent on the guanine nucleotide binding state. It was lower in the GTP conformation, and the mechanism involved the destabilization of the -helical regions of the G-domain by Ecdomain 2.
Keywords: EF-Tu; thermostability; chimeric protein; EF-Tu domains; G-domain; Escherichia coli; Bacillus stearothermophilus
Abbreviations: EF-Tu, elongation factor Tu • EF-1, eukaryotic elongation factor 1 • GST, glutathione S-transferase
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