Local structural features around the C-terminal segment of Streptomyces subtilisin inhibitor studied by the carbonyl carbon nuclear magnetic resonances of three phenylalanyl residues

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Abstract
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.degree. 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. At 50.degree. C, the line shape of the Phe-97 carbonyl carbon appeared to be composed of four poorly separated lines, but they coalesced into a rather broad single line above 60.degree. C. It was thus concluded that the amide hydrogen of Ser-98 is rapidly exchangeable with the solvent hydrogen (or deuterium) at elevated temperatures. The Phe-111 and Phe-113 signals in 50% D2O appeared as double peaks up to 80.degree. C, although the separations of the double peaks were quite different from each other and were 5.0 and 1.7 Hz, respectively. The larger separation observed for Phe-111 was caused by the partial deuteriation (half-deuteriation) of Ala-112 amide nitrogen (the .beta.-shift). A small separation observed for Phe-113 was induced by the partial deuteriation of Phe-113 amide nitrogen (the .gamma.-shift). Since these values of separation were essentially unchanged up to 80.degree. C, we suggest that the local environment around the C-terminus of SSI was hydrophobic and therefore the amide nitrogens of Ala-112 and Phe-113 are inaccessible to the solvent even at elevated temperatures. The results are consistent with the relative solvent accessibilities of these amide nitrogens calculated by using the atomic coordinates obtained by X-ray crystallography. Temperature dependence of the carbonyl chemical shifts indicates that Phe-111 and Phe-113, both of which exist in the hydrophobic interior of SSI, have a considerable degree of motional freedom. The local structure around Phe-97, which exists in the outermost part of the 5-fold .beta.-pleated sheet, seems to be less flexible at lower temperatures but becomes fairly mobile above 60.degree. C. Comparison of the chemical shifts between native and thermally denatured [F]SSI were found to be useful to study the structural characteristics and local environments of the C-terminal segment of the native SSI.