Properties of Truncated Forms of the Elongation Factor 1α from the Archaeon Sulfolobus solfataricus

Abstract

Two truncated forms of the Sulfolohus solfuturicus elongation factor 1 a (SsEF-la), corresponding to the putative domains G+M, Ss(GM)EF-la, and G, Ss(G)EF-la, have been constructed by gene engineering, produced in Escherichin coli and purified. Neither truncated form was able to sustain poly(Phe) synthesis but they were able to bind guanine nucleotides with an affinity much higher with respect to that of the intact factor. However, the difference in the affinity for GDP and GTP became progressively reduced with the extent of the truncation. The values of k, , , and K,,, for GTP of the intrinsic GTPase of SsEF-lu triggered by 3.6 M NaCl were not affected by the deletions. In contrast, both Ss(GM)EF-la and Ss(G)EF-la were less thermostable than the intact factor; the region of the factor most responsible for the loss of resistance against heat inactivation was the C-terminal domain. On the other hand the domain M was the regulator of the thermophilicity of SsEF-la since only Ss(G)EF-la showed a reduced thermophilicity. Remarkably, both Ss(GM)EF-la and Ss(G)EF-la were able to exchange ['HIGDP for GTP at a very high rate so that they were no more sensitive to the stimulatory effect of SsEF-Ij?, which is the nucleotide exchange factor of SsEF-I a.

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

“…The G‐domains set up a “basic level” of the thermal stability of the EF‐Tus (cf. also Sanangelantoni et al 1996; Masullo et al 1997). The level set up by the thermophilic BstG‐domain was ∼20°C higher than that of the EcG‐domain.…”

“…Nock et al (1995) reported that in Thermus thermophilus EF‐Tu, the deletion of domains 2/3 or only of domain 3 provoked a lower affinity for guanine nucleotides similarly as in the E. coli system. In contrast, the same truncation of Sulfolobus solfataricus EF‐1α increased the affinity for GDP and GTP by about one order of magnitude compared with the intact protein (Masullo et al 1997). This implies that the same level of affinity to guanine nucleotides can be attained by different strategies in different EF‐Tus.…”

“…Until now, functional evaluation of individual domains of EF‐Tu has been carried out mainly by the deletion approach, by examining one‐ or two‐domain proteins (Parmeggiani et al 1987; Nock et al 1995; Masullo et al 1997; Cetin et al 1998). In this paper, we address the question of how the functions and thermostability of EF‐Tu from mesophilic Escherichia coli , growing at ∼37°C and EF‐Tu from thermophilic Bacillus stearothermophilus growing at temperatures 55°–60°C are built from the contributions of individual domains.…”

Stability and Function at High Temperature. What Makes a Thermophilic GTPase Different from Its Mesophilic Homologue

“…The EF-Tu of E. coli and EF-1α of S. solfataricus are of similar structure, − both triangular three-domain proteins with a hole in the middle and substantial differences between the reactant GTP (active) and product GDP (inactive) state. Despite the role of interdomain interactions ensuring long-range communication, some essential features of the EF activity can be investigated by considering only the catalytic domain, as the isolated domain is catalytically active. , The catalytic subunit is slightly less thermostable than the three-domain protein for both the mesophile and the thermophile. The inactivation temperature of the catalytic subunit in the EF-Tu is 41 °C as opposed to 46 °C for the entire protein; , for EF-1α, the catalytic subunit loses activity at 84 °C and the entire protein at 94 °C .…”

Differential Contribution of Protein Factors and 70S Ribosome to Elongation

“…The cooperative participation of all three EF-Tu domains is necessary to ensure a phenotype regarding the binding of guanine nucleotides both in T. thermophilus [10] and E. coli [11]. While, for example, high affinity for GDP and GTP is an inherent property of the G-domain in EF-Tu from Bacillus stearothermophilus [12], and partial truncation of Sulfolobus solfataricus EF-1α increases the binding affinity of GDP and GTP by approximately one order of magnitude compared to intact protein [13]. Mutational analysis of catalytic His84 in E. coli EF-Tu and homologous His85 in T. thermophilus EF-Tu revealed similar mechanisms of GTP hydrolysis by these two proteins [14][15][16].…”