LOCAL STRUCTURAL FEATURES AROUND THE C-TERMINAL SEGMENT OF STREPTOMYCES SUBTILISIN INHIBITOR STUDIED BY CARBONYL CARBON NUCLEAR MAGNETIC RESONANCES OF 3 PHENYLALANYL RESIDUES
LOCAL STRUCTURAL FEATURES AROUND THE C-TERMINAL SEGMENT OF STREPTOMYCES SUBTILISIN INHIBITOR STUDIED BY CARBONYL CARBON NUCLEAR MAGNETIC RESONANCES OF 3 PHENYLALANYL RESIDUES
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DOI：
10.1021/bi00378a013
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发表时间：
1987-02-24
期刊：
影响因子：
2.9
通讯作者：
TSUJI, T
中科院分区：
文献类型：
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作者：
KAINOSHO, M;NAGAO, H;TSUJI, T
对枯草杆菌蛋白酶抑制剂（SSI）中苯丙氨酸残基的羰基碳NMR信号进行了选择性分析，发现SSI中所有苯丙氨酸均被[1 - 13 C] Phe标记。[F] SSI光谱中的三个增强共振通过15N，13C双标记技术明确地归属于氨基酸序列中的特定位点。即，在174.9和172.6ppm（在D2O中，pH 7.3，50 ° C）处的共振值分别为1000和1000。C）在[F，GS] SSI和[F，A] SSI的光谱中显示了由于13 C-15 N自旋耦合引起的卫星峰，其中Ser/Gly和Ala残基分别用[15 N] Gly/Ser和[15 N] Ala以及[1 - 13 C] Phe标记。Phe-97和Phe-111的羰基分别与Ser-98和Ala-112的氨基氮形成肽键。这些结果清楚地表明，174.5和172.6 ppm处的信号分别是由于Phe-97和Phe-111。因此，最低场（177.1 ppm）处的信号归属于C-末端Phe-113的羧基碳。利用氘氢交换对苯丙氨酸羰基碳共振线形的影响，研究了苯丙氨酸及其C端侧邻体（Ser-98和Ala-112）的酰胺氢寿命.在此方法中，[F] SSI溶解在50%D_2O（pH7.3）中，在不同温度下的NMR谱测量，氘代同位素位移引起的线型变化进行了分析。在50度。在60 ℃时，Phe-97羰基碳的线形似乎由四条分离不好的线组成，但它们在60 ℃以上合并成一条相当宽的单线。C.因此得出结论，Ser-98的酰胺氢在升高的温度下可与溶剂氢（或氘）快速交换。Phe-111和Phe-113在50%D2O中的信号表现为高达80 °的双峰。C，虽然双峰的分离彼此相当不同，分别为5.0和1.7Hz。对于Phe-111观察到的较大分离是由Ala-112酰胺氮的部分氘代（半氘代）（β-氘代）引起的。移位）。通过Phe-113酰胺氮的部分氘代化诱导了对Phe-113观察到的小分离（γ-移位）。因为这些分离值在高达80 °时基本上不变。C，我们认为SSI的C-末端周围的局部环境是疏水性的，因此Ala-112和Phe-113的酰胺氮即使在升高的温度下也不能接近溶剂。计算结果与用X射线晶体学得到的原子坐标计算的这些酰胺氮的相对溶剂含量一致。羰基化学位移的温度依赖性表明，Phe-111和Phe-113，这两者都存在于SSI的疏水内部，具有相当程度的运动自由度。Phe-97周围的局部结构，其存在于5倍β-半乳糖苷酶的最外部分。褶皱片材在较低温度下似乎柔性较小，但在60 ℃以上变得相当移动的。C.比较天然和热变性[F] SSI之间的化学位移被发现是有用的，以研究天然SSI的C-末端片段的结构特征和局部环境。
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.