Thermostability of multidomain proteins: Elongation factors EF‐Tu from Escherichia coli and Bacillus stearothermophilus and their chimeric forms

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 K d 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 α‐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 α‐helical regions of the G‐domain by Ecdomain 2.