PMID:14691225
| Citation |
Sanderová, H, Hůlková, M, Malon, P, Kepková, M and Jonák, J (2004) Thermostability of multidomain proteins: elongation factors EF-Tu from Escherichia coli and Bacillus stearothermophilus and their chimeric forms. Protein Sci. 13:89-99 |
|---|---|
| Abstract |
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 approximately 20 degrees 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 alpha-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 alpha-helical regions of the G-domain by Ecdomain 2. |
| Links |
PubMed PMC2286523 Online version:10.1110/ps.03272504 |
| Keywords |
Amino Acids/chemistry; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Circular Dichroism; Escherichia coli/chemistry; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Genetic Variation; Geobacillus stearothermophilus/chemistry; Guanosine Diphosphate/metabolism; Guanosine Triphosphate/metabolism; Kinetics; Models, Molecular; Peptide Elongation Factor Tu/chemistry; Peptide Elongation Factor Tu/genetics; Peptide Elongation Factor Tu/metabolism; Protein Binding; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/isolation & purification; Recombinant Fusion Proteins/metabolism; Species Specificity; Temperature |
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