TRANSLATIONAL INITIATION-FACTOR IF2 FROM BACILLUS-STEAROTHERMOPHILUS - A SPECTROSCOPIC AND MICROCALORIMETRIC STUDY OF THE C-DOMAIN

Conformation and stability of the C-terminal domain of initiation fact or IF2 from Bacillus stearothermophilus were analyzed by circular dich roism, fluorescence and Raman spectroscopy, and microcalorimetry under different solvent conditions. From circular dichroism and Raman measu rements, IF2C at neutral pH can be classified as an alpha + beta prote in. Solvent perturbation and Raman spectroscopy indicate a high access ibility of the tyrosine residues in the native protein. The Gdn/HCl-in duced unfolding of IF2C was monitored by circular dichroism. IF2C unfo lding at neutral pH proceeds in two discrete steps. The midpoints (c(m )) and the free energy of unfolding (Delta G(u)(H2O)) Of the first and second transition are 2.05 M and 6.2 kcal . mol(-1) and 4.1 M and 12. 9 kcal . mol(-1), respectively. ANS does not bind to the stable interm ediate formed at 3 M Gdn/HCl. It seems likely that IF2C is composed of two subdomains which unfold in a stepwise process. Melting experiment s at pH 7.0 are impaired by irreversible aggregation at higher tempera tures. However, in Gdn/HCl containing buffer at denaturant concentrati ons up to 1.5 M the melting becomes a reversible process and can be an alyzed by differential scanning calorimetry. At Gdn/HCl concentrations between 1.0 and 1.5 M, IF2C seems to be composed of two folding units with T-m values of about 60 and 78 degrees C and folding enthalpy val ues (Delta H-m) of about 37 and 58 kcal . mol(-1). At pH values below pH 3.0, IF2C can adopt a new acid-induced conformation, which is chara cterized by a high secondary structure content and a strong ANS bindin g. The Gdn/HCl-induced unfolding of IF2C at pH 2.6 takes place only in one discrete step with a midpoint c, of 3.3 M and a Delta G(AUa)(H2O) , of 11.9 kcal . mol(-1).