Determining secondary structure in spider dragline silk by carbon-carbon correlation solid-state NMR spectroscopy
Determining secondary structure in spider dragline silk by carbon-carbon correlation solid-state NMR spectroscopy
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DOI：
10.1021/ja8021208
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发表时间：
2008-07-30
影响因子：
15
通讯作者：
Yarger, Jeffery L.
中科院分区：
文献类型：
--
作者：
Holland, Gregory P.;Creager, Melinda S.;Yarger, Jeffery L.
采集了棒形新丝蛛富碳牵引丝纤维的二维(2D)C-13-C-13相关谱。在快速磁角旋转(MAS)和偶极辅助旋转共振(DAR)重耦合下获得了二维核磁共振谱，以增强C-13自旋之间的磁化传递。利用重新耦合周期短(150ms)的光谱来提取丝绸光谱中每个标记氨基酸的所有碳共振的不同化学位移，从而得到完整的共振归属。本文给出的核磁共振结果首次允许提取蜘蛛丝中每种C-13标记氨基酸的精确的化学位移值。利用重新耦合周期较长的光谱(S 1)检测相邻氨基酸之间的分子间磁化交换。这一信息被用来将核磁共振共振归因于蜘蛛丝蛋白中普遍存在的特定重复氨基酸基序。这些结果表明，甘氨酸和丙氨酸都存在于两种不同的结构环境中：无序的31螺旋构象和有序的P-折叠结构。前者属于Gly-Gly-Ala基序，而后者属于聚(Ala)和聚(Gly-Ala)结构域。
Two-dimensional (2D) C-13-C-13 NMR correlation spectra were collected on C-13-enriched dragline silk fibers produced from Nephila clavipes spiders. The 2D NMR spectra were acquired under fast magicangle spinning (MAS) and dipolar-assisted rotational resonance (DARR) recoupling to enhance magnetization transfer between C-13 spins. Spectra obtained with short (150 ms) recoupling periods were utilized to extract distinct chemical shifts for all carbon resonances of each labeled amino acid in the silk spectra, resulting in a complete resonance assignment. The NMR results presented here permit extraction of the precise chemical shift of the carbonyl environment for each C-13-labeled amino acid in spider silk for the first time. Spectra collected with longer recoupling periods (1 s) were implemented to detect intermolecular magnetization exchange between neighboring amino acids. This information is used to ascribe NMR resonances to the specific repetitive amino acid motifs prevalent in spider silk proteins. These results indicate that glycine and alanine are both present in two distinct structural environments: a disordered 31-helical conformation and an ordered P-sheet structure. The former can be ascribed to the Gly-Gly-Ala motif while the latter is assigned to the poly(Ala) and poly(Gly-Ala) domains.