COLD DENATURATION AND HEAT DENATURATION OF STREPTOMYCES SUBTILISIN INHIBITOR .2. H-1-NMR STUDIES

Structural transitions of the protein Streptomyces subtilisin inhibitor (SSI) from the native state to the cold-denatured and heat-denatured states were studied by H-1 NMR spectroscopy in the temperature range from -10 to 60-degrees-C in the acidic pH range. Assignments of some of the H-1 NMR signals of SSI in the cold-denatured and heat-denatured states were performed by a combined use of selective deuteration and site-directed mutagenesis. Throughout the pH range from 2.1 to 3.1, both transitions were cooperative and basically only three distinct spectra corresponding to structures in the cold-denatured, native, and heat-denatured states were detected. In the cold-denatured state, the side-chain signals of Met73, His106, at least one Val, and two Leu were observed at distinctly shifted positions from those for a random-coiled structure, suggesting the formation of a tertiary structure, while those of Met70, His43, and Ala2 were observed at positions for a random-coiled structure. This tertiary structure in the cold-denatured state is entirely different from that in the native state, as some amino acid residues exposed to the solvent in the native state (e.g., Met73, His106) are buried while those sequestered in the native state (e.g., His43) are exposed. In the heat-denatured state, however, most H-1 NMR signals were observed at random-coiled positions, indicating that there is much less tertiary structure in the heat-denatured state than in the cold-denatured state. At pH values below 2.09, a structural transition was observed from the cold-denatured state to the heat-denatured state without passing through the native state. A sedimentation equilibrium experiment indicated that the two subunits of SSI were dissociated in both the heat-denatured and cold-denatured states, eliminating the possibility of formation of a tertiary structure in the cold-denatured state by intersubunit interaction, such as by aggregation.