E¡ect of N-domain on the stability of elongation factor Ts from Thermus thermophilus Eva Valus -ova ¨ a ; 1 , Erik Sedla ¨k a ; 1 , Maria ¨n Antal| ¨k a;b , Ste¡en Nock c ; 2 , Mathias Sprinzl c ; * a Department of Biochemistry, Faculty of Science P.J. S í afa ¨rik University, Kos -ice, Slovak Republic b Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kos -ice, Slovak Republic c Laboratorium fu «r Biochemie, Universita «t Bayreuth, D-95440 Bayreuth, Germany Received 20 December 2000; received in revised form 23 February 2001; accepted 26 February 2001 Abstract 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 V13. 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. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Domain protein; Thermal stability; Elongation factor Ts; Thermophile; Cooperative unit 1. Introduction Elongation factor Ts (EF-Ts) functions as a nucle- otide-exchange factor by binding elongation factor TuWGDP complex (EF-TuWGDP) and accelerates the GDP dissociation. Thus, EF-Ts promotes the transi- tion of EF-Tu from the inactive GDP form to the active GTP form. Thermus thermophilus EF-Ts forms a homodimer in solution [1,2]. Proteolytic cleavage experiment [2] and structure analysis [3] revealed a two domain structure of T. thermophilus EF-Ts: N- 0167-4838 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII:S0167-4838(01)00172-8 Abbreviations : EF-Ts, elongation factor Ts ; EF-Ts(C190A), EF-Ts with cysteine 190 replaced for alanine; EF-Tu, elongation factor Tu; DSC, di¡erential scanning calorimetry; CD, circular dichroism * Corresponding author. Fax: +49-921-552432; E-mail : mathias.sprinzl@uni-bayreuth.de 1 Both authors contributed equally to this work. 2 Present address : Zyomyx, Inc., Hayward, CA, USA. Biochimica et Biophysica Acta 1547 (2001) 117^126 www.bba-direct.com