Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR
Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR
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DOI：
10.3791/59152
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发表时间：
2019-02-01
影响因子：
1.2
通讯作者：
Wang, Tuo
中科院分区：
文献类型：
--
作者：
Kirui, Alex;Widanage, Malitha C. Dickwella;Wang, Tuo
该协议展示了如何均匀地生产 C-13、N-15 标记的真菌材料，以及如何对这些软材料进行固态 NMR 和灵敏度增强的 DNP 实验。还详细介绍了植物生物质的样品处理程序。该方法可以测量一系列一维和二维 C-13-C-13/N-15 相关光谱，从而能够以最小的扰动对原始状态的复杂生物材料进行高分辨率结构阐明。同位素标记可以通过量化一维光谱中的强度和二维相关光谱中的偏振转移效率来检查。动态核极化 (DNP) 样品制备的成功可以通过灵敏度增强因子来评估。进一步的实验检查多糖和蛋白质的结构方面将产生三维结构的模型。这些方法可以进行修改和调整，以研究各种富含碳水化合物的材料，包括植物、真菌、藻类和细菌的天然细胞壁，以及合成或设计的碳水化合物聚合物及其与其他分子的复合物。
This protocol shows how uniformly C-13, N-15-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D C-13-C-13/N-15 correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.