Effect of N-domain on the stability of elongation factor Ts from Thermus thermophilus

Elongation factor Ts (EF-Ts) from Thermus thermophilus forms a stable, func tionally active homodimer in solution. Its monomer is composed of two domai ns: amino-terminal domain containing 50 amino acid residues and a larger, 1 46 residues long, C-domain which participates in dimerization of EF-Ts. Eff ect of removal of the N-domain on the conformational stability of EF-Ts has been studied. For comparison, the stabilities of both the full-length EF-T s and its C-domain were studied by differential scanning calorimetry, elect ronic absorption and fluorescence spectroscopies over a pH range from 4 to similar to 13. Thermal denaturation of EF-Ts and of C-domain, followed by c ircular dichroism at 222 nm, at pH 7.0, and the pH dependence of the fluore scence of the single tryptophan 30 residue indicate a conformational instab ility of the N-domain. While N-domain does not affect the stability of full -length EF-Ts at acidic pH, its removal leads to stabilization of the rest of the protein at basic pH. This is reflected by higher values of transitio n temperatures and calorimetric enthalpies of C-domain as compared to the f ull-length EF-Ts. High mobility of the N-domain in alkaline pH conditions d ecreased the thermal stability of covalently linked C-domain of EF-Ts. An i ncrease in intramolecular interactions at acidic pH together with a decreas e of conformational entropies of the thermally denatured proteins most like ly diminishes this destabilization effect. (C) 2001 Elsevier Science B,V. A ll rights reserved.