JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
Results
Discussion
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Acknowledgements
Materials
References
자동 번역
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생화학

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

Published: February 12, 2019
doi:
10.3791/59152
, , , , ,
1Department of Chemistry,Louisiana State University, 2National High Magnetic Field Laboratory, 3Departments of Pediatrics, and Microbiology, Immunology and Parasitology,Louisiana State University Health Sciences Center

Summary

提出了一种用于多维固态核磁共振光谱和动态核极化 (dnp) 研究的 13 c、15个n 标记真菌和植物样品的制备方案。

Abstract

该协议显示了如何均匀地生产13c, 15n 标记的真菌材料, 以及如何进行这些软材料的固态核磁共振和敏感增强的 dnp 实验。并对植物生物质的样品处理工艺进行了详细的介绍。这种方法允许测量一系列的一维和 2d 13c-13 cp-15 n相关光谱, 从而能够以最小的扰动对其原生状态下的复杂生物材料进行高分辨率结构描述.通过量化一维光谱中的强度和二维相关谱中的偏振传递效率, 可以检测同位素标记。动态核极化 (dnp) 样品制备的成功与否可以用灵敏度增强因子来评价。进一步的实验, 研究多糖和蛋白质的结构方面将导致一个三维架构模型。这些方法可以进行修改和调整, 以研究广泛的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物质的复合物。分子。

Introduction

碳水化合物在各种生物过程中发挥着核心作用, 如储能、结构建筑、细胞识别和粘附。它们在细胞壁中被丰富, 细胞壁是植物、真菌、藻类和细菌的基本成分 1,2,3.细胞壁是生产生物燃料和生物材料的核心来源, 也是抗菌疗法4、5、6、7、8的一个有希望的目标,9个。

几十年来, 人们一直在努力利用四种主要的生化或遗传方法进行结构表征, 从而大大促进了对这些复杂材料的当代理解。第一种主要方法依靠顺序处理使用苛刻的化学物质或酶将细胞壁分解成不同的部分, 然后对每 10分的糖进行成分和连锁分析。这种方法揭示了聚合物的域分布, 但由于生物分子的化学和物理性质, 解释可能具有误导性。例如, 很难确定碱萃取分数是来自结构较少的分子的单个域, 还是来自具有可比溶解性的空间分离分子。其次, 提取的部分或整个细胞壁也可以测量使用溶液核磁共振, 以确定共价键, 也称为交联, 不同分子之间的 11,12,13, 14,15。通过这种方法, 可以探讨共价锚杆的详细结构, 但由于多糖的溶解度较低、交联位点数量相对较少以及对稳定的非共价效应的无知, 可能会存在局限性。多糖填料, 包括氢键、范德华力、静电相互作用和聚合物纠缠。第三, 利用分离的多糖16、17、18、19 在体外测定了结合亲和力, 但纯化过程可能会发生重大变化。这些生物分子的结构和性质。这种方法也不能复制复杂的沉积和组装的大分子后的生物合成。最后, 某些细胞壁成分减毒产生的遗传突变体的表型、细胞形态和力学性质揭示了多糖的结构功能, 但需要更多的分子证据来弥补这些因素的联系。宏观观测与蛋白质机械的工程功能 20。

近年来在多维固态核磁共振光谱的发展和应用方面取得的进展为解决这些结构难题提供了一个独特的机会。通过2d/3d 固态核磁共振实验, 可以在不产生重大扰动的情况下, 对富含碳水化合物材料的成分和结构进行高分辨率研究。对植物的原生和二级细胞壁、经过催化处理的生物量、细菌生物膜、真菌中的色素鬼以及最近作者在病原真菌中的完整细胞壁进行了结构研究。熏蒸曲霉21,22,23,24,25,26,27,28,29,30,31. 动态核两极分化的发展 32 、33、34、35、36、37、38,39,40,41,42极大地促进了核磁共振结构的阐明, 因为 dnp 的灵敏度增强明显缩短了这些复杂生物材料的实验时间。这里描述的协议详细介绍了同位素标记真菌a. 熏蒸剂的程序, 以及为固态核磁共振和 dnp 表征准备真菌和植物样品的程序。类似的标签程序应适用于其他培养基改变的真菌, 样品制备程序应普遍适用于其他富含碳水化合物的生物材料。

Protocol

1.生长13c, 15n 标记的富米达斯下酯类液体培养基 未标记和13c、 15n 标记生长介质的制备注: 酵母提取物丙酮葡萄糖培养基 (ypd) 和改进的最小培养基43均用于维持真菌培养。高压灭菌后的所有步骤都是在层流罩中执行的, 以最大限度地减少污染。 无标记液体介质的制备: 在100毫升蒸馏水中溶解6.5 克 ypd 粉末, 在134°c 下高压灭菌2…

Representative Results

同位素标记大大提高了核磁共振灵敏度, 并使测量一系列 2d 13c-13 c 和13c-15n 相关光谱的可能性成为可能, 以分析其组成、水化、流动性和包装。聚合物, 它将被集成来构建细胞壁结构的三维模型 (图 1)。如果统一的标签成功, 可以在1小时内收集一套完整的 1d13c和 15n 光谱, 每个标准的2d 光谱测量时间不应?…

Discussion

与生化方法相比, 固态核磁共振作为一种无损、高分辨率的技术具有优势。核磁共振在成分分析中也是定量的, 与大多数其他分析方法不同的是, 不存在生物聚合物溶解度有限所带来的不确定性。目前协议的建立有助于今后对富含碳水化合物的生物材料和功能化聚合物的研究。然而, 应当指出, 共振分配和数据分析可能很耗时, 通常需要系统的培训。作者目前正在开发工具和数据库, 以帮助没有经验?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家科学基金会通过 nsf oia-1833040 提供的支持。国家高磁场实验室 (nhmfl) 由国家科学基金会通过 dmr-117490 和佛罗里达州提供支助。nhmfl 的 mas-dnp 系统部分由 nih s10 od018519 和 nsf che-1229170 供资。

Materials

Ammonium Molybdate Tetrahydrate Acros Organics 12054-85-2
AMUPol Cortecnet C010P002
Analytical weighing balance Ohaus B730439218 Model PA84C
Bioclave 16 L VWR 470230-598
Biosafety Cabinet Labconco corporation 302319100
Boric acid VWR BDH9222 store at 15-30 °C
Cobalt(II) Chloride Hexahydrate Honeywell|Fluka 60820 ≥98 %
Copper(II) Sulfate Pentahydrate BDH BDH9312 ≥98 %
Corning LSE shaking incubator Thermo Fisher Scientific 7202152
D2O Sigma Aldrich 151882 99.9 atom % D
d6-DMSO Sigma Aldrich 151874 99.9 atom % D
d8-glycerol Sigma Aldrich 447498 ≥99 atom % D
Dialysis tubing 3.2 kDa Sigma Aldrich D2272 132724
Dipotassium Phosphate VWR BDH9266 ≥98 %
Glycerol Sigma Aldrich G5516 ≥99.5 %
Heraus Megafuge 16R Centrifuge Thermo Fischer Scientific 750004271 Maximum RCF 25,830 x g
HR-MAS Disposable Insert Kit Bruker B4493 Kel-F
Iron(II) Sulfate Heptahydrate Alfa Aesar 14498 ≥99+ %
Magnesium Sulfate Heptahydrate VWR 10034998 store at 18-26 °C
Manganese(II) Chloride Tetrahydrate Alfa Aesar 11563 ≥99 %
Monopotassium Phosphate VWR 470302-254 ≥99 %
pH Meter Mettler Toledo B706689216
Tetrasodium Ethylenediaminetetraacetate Acros Organics 13235-36-9 ≥99.5 %
Zinc Sulfate Heptahydrate Alfa Aesar 33399 ≥98 %
12C3, d8-glycerol Cambridge Isotope Laboratory CDLM-8660 12C3, 99.95%; D8, 98%
13C6-glucose Sigma Alrdrich 364606 ≥99 % (CP)
15N-sodium nitrate Sigma Aldrich 364606 ≥98 % 15N, ≥99 (cp)
3.2 mm sapphire NMR rotor Cortecnet B6939
3.2 mm Silicone plug Bruker B7089
4 mm MAS Rotor Kit Bruker H14355 Zirconia
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Tags

Fungal MaterialsPlant MaterialsSolid-state NMRDynamic Nuclear PolarizationBiomaterialsCarbohydratesFungal Cell WallsCell CultureYPD MediumLabeled Minimal MediumIsotope LabelingCarbon-13Nitrogen-15SterilizationAutoclaveCell Culture Techniques

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Kirui, A., Dickwella Widanage, M. C., Mentink-Vigier, F., Wang, P., Kang, X., Wang, T. Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR. J. Vis. Exp. (144), e59152, doi:10.3791/59152 (2019).
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