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, functionally active homodimer in solution. Its monomer is composed of two domains: amino-terminal domain containing 50 amino acid residues and a larger, 146 residues long, C-domain which participates in dimerization of EF-Ts. Effect 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-Ts and its C-domain were studied by differential scanning calorimetry, electronic absorption and fluorescence spectroscopies over a pH range from 4 to approximately 13. Thermal denaturation of EF-Ts and of C-domain, followed by circular dichroism at 222 nm, at pH 7.0, and the pH dependence of the fluorescence of the single tryptophan 30 residue indicate a conformational instability 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 transition temperatures and calorimetric enthalpies of C-domain as compared to the full-length EF-Ts. High mobility of the N-domain in alkaline pH conditions decreased the thermal stability of covalently linked C-domain of EF-Ts. An increase in intramolecular interactions at acidic pH together with a decrease of conformational entropies of the thermally denatured proteins most likely diminishes this destabilization effect.
Valusová
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E
Department of Biochemistry, Faculty of Science P.J. Safarik University, Slovak Republic.
Sedlák
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E
Antalík
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M
Nock
S
S
Sprinzl
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M
D002152:Calorimetry, Differential Scanning; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000737:chemistry / Q000378:metabolism*
D010445:Peptide Elongation Factors; C023512:elongation factor Ts