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. Study holds ProTherm entries: 16803, 16804, 16805, 16806, 16807, 16808, 16809, 16810, 16811, 16812, 16813, 16814, 16815, 16816 Extra Details: Protein in GDP conformation. 10 mM 2-mercaptoethanol was added in the experiment. EF-Tu; thermostability; chimeric protein; EF-Tu domains; G-domain; Escherichia coli; Bacillus stearothermophilus
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:48 p.m.
|Number of data points||14|
|Proteins||Elongation factor Tu 1 ; Elongation factor Tu ; Elongation factor Tu 1|
|Assays/Quantities/Protocols||Experimental Assay: Tm details:Additives 2-mercaptoethanol (3.10 mM), ; Experimental Assay: Tm details:Additives 2-mercaptoethanol (10 mM),|
|Libraries||Mutations for sequence MAKAKFERTKPHVNIGTIGHVDHGKTTLTAAITTVLAKQGKAEAKAYDQIDAAPEERERGITISTAHVEYETEARHYAHVDCPGHADYVKNMITGAAQMDGAILVVSAADGPMPQTREHILLSRQVGVPYIVVFLNKCDMVDDEELLELVEMEVRDLLSEYDFPGDEVPVIKGSALKALEGDPKWEEKIIELMNAVDEYIPTPQREVDKPFMMPIEDVFSITGRGTVATGRVERGTLKVGDPVEIIGLSDEPKATTVTGVEMFRKLLDQAEAGDNIGALLRGVSRDEVERGQVLAKPGSITPHTKFKAQVYVLTKEEGGRHTPFFSNYRPQFYFRTTDVTGIITLPEGVEMVMPGDNVEMTVELIAPIAIEEGTKFSIREGGRTVGAGSVSEIIE ; Mutations for sequence SKEKFERTKPHVNVGTIGHVDHGKTTLTAAITTVLAKTYGGAARAFDQIDNAPEEKARGITINTSHVEYDTPTRHYAHVDCPGHADYVKNMITGAAQMDGAILVVAATDGPMPQTREHILLGRQVGVPYIIVFLNKCDMVDDEELLELVEMEVRELLSQYDFPGDDTPIVRGSALKALEGDAEWEAKILELAGFLDSYIPEPERAIDKPFLLPIEDVFSISGRGTVVTGRVERGIIKVGEEVEIVGIKETQKSTCTGVEMFRKLLDEGRAGENVGVLLRGIKREEIERGQVLAKPGTIKPHTKFESEVYILSKDEGGRHTPFFKGYRPQFYFRTTDVTGTIELPEGVEMVMPGDNIKMVVTLIHPIAMDDGLRFAIREGGRTVGAGVVAKVLS|