NMR OF SILK FIBROIN .3. ASSIGNMENT OF CARBONYL CARBON RESONANCES AND THEIR DEPENDENCE ON SEQUENCE AND CONFORMATION IN BOMBYX-MORI SILK FIBROIN USING SELECTIVE ISOTOPIC LABELING
NMR OF SILK FIBROIN .3. ASSIGNMENT OF CARBONYL CARBON RESONANCES AND THEIR DEPENDENCE ON SEQUENCE AND CONFORMATION IN BOMBYX-MORI SILK FIBROIN USING SELECTIVE ISOTOPIC LABELING
复制标题
DOI：
10.1021/ma00141a036
复制
发表时间：
1984-01-01
期刊：
影响因子：
5.5
通讯作者：
ITOH, T
中科院分区：
文献类型：
--
作者：
ASAKURA, T;WATANABE, Y;ITOH, T
用13C NMR对家蚕丝素进行了序列和构象分析。丝素蛋白光谱中羰基区有16个峰。通过比较[1-13C] ala标记的、[l-13C] gly标记的和未富集的丝素蛋白的光谱，将这些峰划分为5种氨基酸残基的羰基碳。[15N] gly标记的丝蛋白羰基区13C-15N直接自旋耦合产生的卫星峰表明存在X- gly序列，其中X= Ala或Ser残基。进一步的详细分配是通过比较丝素蛋白的结晶部分，即沉淀部分，在胰凝乳酶水解丝素蛋白。大多数峰值被分配给主序列（文本中的表1）。在Ala和Gly残基的羰基碳中，序列引起的化学位移的分散性高达0.7 ppm。此外，根据水溶液中分配给Gly-Ala-或Gly-Ser-序列的Gly羰基共振的远程耦合常数3JC/-nc>-h，确定了Ala和Ser残基NC键周围的内旋角。利用[l-13C] gly标记的丝素蛋白和仔细的核磁共振观察，可以直接确定丝素蛋白的耦合常数，实验误差约为0.1 Hz。在对3JC'-n-ca-H的重偶线间距进行校正后，其值与氧化氘中Ac-Ala-NDMe和Ac-Ser-NDMe的相应耦合值基本一致。这些事实使我们得出结论：随机螺旋丝蛋白主链的柔韧性主要来自于水溶液中与碳相邻的NC”和C”-C”单键旋转的可能性。
The sequentialand conformational analyses of Bombyx mori silkfibroin have been performed by 13C NMR spectroscopy. Sixteen peaks were observed in the carbonyl region of the silkfibroin spectrum. These peaks were classified into the carbonylcarbons of five kinds of amino acid residues from a comparison of the spectraamong [1-13C] Ala-labeled,[l-13C] Gly-labeled, and unenriched silk fibroins. The observation of satellite peaks due to the 13C-15N direct spin coupling in the carbonyl region of [15N] Gly-labeled fibroin indicated the presence of the-X-Gly-sequence, where X= Ala or Ser residue. A further detailed assignment was performed from a comparison of the silkfibroin spectrum with that of the crystalline fraction, ie, the precipitated fraction, after chymotrypsin hydrolysis of the fibroin. Most of the peaks were assigned to the primary sequence (Table I in the text). The dispersions of the chemical shifts due to sequence were as large as 0.7 ppm for the carbonyl carbons of Ala and Gly residues. In addition, the internal rotation angles,, around the NC “bonds of the Ala and Ser residues were determined from the long-range coupling constant 3JC/-nc>-h in the Gly carbonyl resonances assigned to the-Gly-Ala-or-Gly-Ser-sequences in the aqueous solution. The use of [l-13C] Gly-labeled silk fibroin and careful NMR observation made it possible to determine the coupling constant directlywith an experimental error of ca. 0.1 Hz for silk protein. After correcting 3JC'-n-ca-H for the doublet spacings in the case of overlapping components, thevalues were in fair agreement with the corresponding coupling values for Ac-Ala-NDMe and Ac-Ser-NDMe in deuterium oxide. These facts lead to the conclusion that the flexibility of the main chain of random coil fibroin originates essentially from the possibilities of rotation about the NC “and C “-C'single bonds adjacent to the-carbons in aqueous solution.