Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 2. 1H 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 1H NMR spectroscopy in the temperature range from -10 to 60 degrees C in the acidic pH range. Assignments of some of the 1H 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 1H 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Tamura
A
A
Department of Chemistry, Faculty of Science, Kyoto University, Japan.
Kimura
K
K
Akasaka
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D003080:Cold Temperature; D006358:Hot Temperature; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*
D001426:Bacterial Proteins; D015842:Serine Proteinase Inhibitors; C018150:subtilisin inhibitor protein, Streptomyces; D006859:Hydrogen