Local structural features around the C-terminal segment of Streptomyces subtilisin inhibitor studied by carbonyl carbon nuclear magnetic resonances three phenylalanyl residues.
The carbonyl carbon NMR signals of the Phe residues in Streptomyces subtilisin inhibitor (SSI) were selectively observed for [F]SSI, in which all phenylalanines were uniformly labeled with [1-13C]Phe. The three enhanced resonances in the spectrum of [F]SSI were unambiguously assigned to the specific sites in the amino acid sequence by means of 15N,13C double-labeling techniques. Namely, the resonances at 174.9 and 172.6 ppm (in D2O, pH 7.3, 50 degrees C) showed the satellite peaks due to 13C-15N spin coupling in the spectra of [F,GS]SSI and [F,A]SSI, in which Ser/Gly and Ala residues were labeled with [15N]Gly/Ser and [15N]Ala, respectively, together with [1-13C]Phe. The carbonyl groups of Phe-97 and Phe-111 are involved in peptide bonds with the amino nitrogens of Ser-98 and Ala-112, respectively. These results clearly indicate that the signals at 174.5 and 172.6 ppm are due to Phe-97 and Phe-111, respectively. The signal at the lowest field (177.1 ppm) was thus assigned to the carboxyl carbon of the C-terminal Phe-113. The lifetimes of the amide hydrogens of the three Phe residues and their C-terminal-side neighbors (Ser-98 and Ala-112) were investigated by using the effect of deuterium-hydrogen exchange of amide on the line shapes (DEALS) for the Phe carbonyl carbon resonances. In this method, the NMR spectra of [F]SSI dissolved in 50% D2O (pH 7.3) were measured at various temperatures, and the line shape changes caused by deuteriation isotope shifts were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010649:Phenylalanine; D011480:Protease Inhibitors; D011487:Protein Conformation; D013816:Thermodynamics
D001426:Bacterial Proteins; D011480:Protease Inhibitors; C018150:subtilisin inhibitor protein, Streptomyces; D010649:Phenylalanine