Nucleotide binding and GTP hydrolysis by elongation factor Tu from Thermus thermophilus as monitored by proton NMR

Abstract

Proton NMR experiments of the GTP/GDP-binding protein EF-Tu from the extremely thermophilic bacterium Thermus thermophilus HB8 in H2O have been performed paying special attention to the resonances in the downfield region (below 10 ppm). Most of these downfield signals are due to hydrogen bonds formed between the protein and the bound nucleotide. However, three downfield resonances appear even in the nucleotide-free EF-Tu. The middle and C-terminal domain (domain II/III) of EF-Tu lacking the GTP/GDP-binding domain gives rise to an NMR spectrum that hints at a well-structured protein. In contrast to native EF-Tu, the domain II/III spectrum contains no resonances in the downfield region. Several downfield resonances can be used as a fingerprint to trace hydrolysis of protein-bound GTP and temperature effects on the EF-Tu.GDP spectra. NMR studies of the binding of guanosine nucleotide analogues (GMPPNP, GMPPCP) to nucleotide-free EF-Tu have been carried out. The downfield resonances of these complexes differ from the spectrum of EF-Tu.GTP. Protected and photolabile caged GTP was bound to EF-Tu, and NMR spectra before and after photolysis were recorded. The progress of the GTP hydrolysis could be monitored using this method. The downfield resonances have been tentatively assigned taking into account the known structural and biochemical aspects of EF-Tu nucleotide-binding site.

Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis

“…His-84 of EF-Tu has been proposed to activate a water molecule for nucleophilic attack on the ␥-phosphate by extracting one of its protons (3,4). This proposal agrees with studies identifying His-84 as the catalytic residue that stabilizes the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or the ␥-phosphate group of GTP (5).…”

Identification and Purification of Translation Initiation Factor 2 (IF2) from Thermus thermophilus

“…glycerol (standard buffer). Growth of 7: tlzermophilus, cell disruption and preparation of a Sl00 supernatant were performed as described (Limmer et al, 1992). Ribosomes in the pellet of the Sl00 preparation were suspended in standard buffer with 1 M NH,CI, and the tube was continuously inverted for 10 h. After centrifugation at 100000Xg for 3 h, the supernatant was withdrawn and dialyzed against standard buffer (high salt ribosomal wash).…”

Discrimination against misacylated tRNA by chloroplast elongation factor Tu

“…EF-Tu from E. coli was purified as described [20]. The protein concentration of all enzyme preparations was determined according to Whitaker and Granum [21].…”