UREA-INDUCED CONFORMATIONAL-CHANGES IN COLD-DENATURED AND HEAT-DENATURED STATES OF A PROTEIN, STREPTOMYCES SUBTILISIN INHIBITOR

Streptomyces subtilisin inhibitor (SSI) is known to exist in at least two distinct denatured states, cold-denatured (D') and heat-denatured (D) under acidic conditions. In the present work, we investigated the manner how increasing urea concentration from 0 to 8 M changes the pol ypeptide chain conformation of SSI that exists initially in the D' and D states as well as in the native state (N), in terms of the secondar y structure, the tertiary structure, and the chain form, based on the results of the experiments using circular dichroism (CD), small-angle X-ray scattering (SAXS) and H-1-NMR spectroscopy. Our results indicate that the urea-induced conformational transitions of SSI under typical conditions of D' (pH 1.8, 3 degrees C) occur at least in two steps. I n the urea concentration range of 0-2 M (step 1), a cooperative destru ction of the tertiary structure occurs, resulting in a mildly denature d state (D-U), which may still contain a little amount of secondary st ructures. In the concentration range of 2-4 M urea (step 2), the D-U s tate gradually loses its residual secondary structure, and increases t he radius of gyration nearly to a maximum value. At 4 M urea, the poly peptide chain is highly disordered with highly mobile side chains. Inc reasing the urea concentration up to 8 M probably results in the more highly denatured or alternatively the stiffer chain conformations. The conformational transition starting from the N state proceeds essentia lly the same way as in the above scheme in which D' is replaced with N . The conformational transition starting from the D state lacks step 1 because the D state contains no tertiary structures and is similar to the D-U state. The fact that similar conformations are reached at ure a concentrations above 2 M from different conformations of D', D, and N indicates that the effect of urea dominates in determining the polyp eptide conformation of SSI in the denatured states rather than the pH and temperature.