Lamberti, Anna (2013) INTERACTION OF EUBACTERIAL LIGANDS WITH ARCHAEAL ELONGATION FACTOR 1α: MODULATION OF ITS MOLECULAR AND FUNCTIONAL PROPERTIES. [Tesi di dottorato]

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Abstract

The interaction between the archaeal elongation factor 1α from Sulfolobus solfataricus (SsEF–1α) and some eubacterial ligands, such as eubacterial antibiotics, tetracycline and pulvomycin, and the modified guanosine nucleotide ppGpp was investigated. SsEF–1α had already been characterised for its structural and functional properties in the laboratory where the research work was carried out. This ubiquitous protein presents structural properties more similar to the eubacterial counterpart and functional propertis more similar to the eukaryal ones. Tetracycline and pulvomycin are eubacterial antibiotics that inhibit protein synthesis. Tetracycline prevents the binding of aa–tRNA to the A site of the ribosome, whereas pulvomycin inhibits the formation of the ternary complex between the elongation factor Tu (EF–Tu), GTP and aa–tRNA. The interaction between SsEF–1α and these antibiotics was demonstrated either by fluorescence spectroscopy in the aromatic region of the spectrum of this protein or by the effects produced by the antibiotic on some functional properties of the elongation factor. The results indicated that both antibiotics were able to inhibit archaeal protein synthesis and that pulvomycin, but not tetracycline, was able to reduce the affinity of SsEF–1α for aa–tRNA in presence of GTP. Moreover, tetracycline produced an increase in GDP/GTP exchange rate and a slight inhibition of the intrinsic GTPase; vice versa, in the presence of pulvomycin, the exchange rate was higher for both guanosine nucleotides and a stimulation of GTPase activity was observed. Furthermore, the interaction between SsEF–1α and tetracycline caused a reduction of stability to the heat treatment, whereas pulvomycin exerted a protective effect on the elongation factor against chemical denaturation. Regarding fluorescence spectroscopy, in the presence of tetracycline an increase in the quantum yield in the fluorescence spectrum of SsEF–1α was observed. A similar behaviour was also observed for Ss(GM)EF– 1α but not for Ss(G)EF–1α, two engineered forms of the elongation factor; these results indicated that the M–domain is essential for the interaction with tetracycline. Pulvomycin, instead, exerted a strong fluorescence quenching at λmax which was accompanied by the appearance of a new peak at 360 nm for all proteins analysed, suggesting that the intact form of SsEF–1α is required for the interaction. ppGpp, also called "magic spot I", is a molecule involved in the “Stringent Control” both in eubacteria and plants during amino acid starvation. It is a compound structurally similar to GDP, characterised by the presence of an additional diphosphate group, bound through a phosphoester bond to the oxygen in position 3’ of ribose. The interaction between SsEF–1α and this molecule was investigated because ppGpp was found bound to a recombinant form of the elongation factor, used for crystallographic studies. ppGpp was able to inhibit in vitro protein synthesis; the affinity of SsEF–1α for aa–tRNA in presence of the nucleotide tetra–phosphate resulted intermediate between that for GDP and GTP. Moreover, ppGpp reduced the affinity of SsEF–1α for guanosine nucleotides through a competitive mechanism. Regarding the effect on the intrisic GTPase, the presence of ppGpp caused an increase of Km with any variation in kcat, indicating also in this case a competitive inhibition behaviour. Finally, the presence of the nucleotide tetra–phosphate rendered SsEF–1α more stable to heat treatement. In conclusion, the data presented demonstrated an interaction between the archaeal elongation factor 1α from Sulfolobus solfataricus and some eubacterial ligands. These results could be used as probe to investigate phylogenetic relationship among living domain.

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